CN100488562C - Coagulation factor VII or VIIa-like molecules - Google Patents

Coagulation factor VII or VIIa-like molecules Download PDF

Info

Publication number
CN100488562C
CN100488562C CNB018048641A CN01804864A CN100488562C CN 100488562 C CN100488562 C CN 100488562C CN B018048641 A CNB018048641 A CN B018048641A CN 01804864 A CN01804864 A CN 01804864A CN 100488562 C CN100488562 C CN 100488562C
Authority
CN
China
Prior art keywords
polypeptide
conjugate
amino acid
fviia
fvii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB018048641A
Other languages
Chinese (zh)
Other versions
CN1400910A (en
Inventor
金·V·安德森
安德斯·H·佩德森
克劳斯·博尼斯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maxygen Holdings Ltd
Original Assignee
Maxygen Holdings Ltd
Bayer Healthcare LLC
Maxygen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Maxygen Holdings Ltd, Bayer Healthcare LLC, Maxygen Inc filed Critical Maxygen Holdings Ltd
Publication of CN1400910A publication Critical patent/CN1400910A/en
Application granted granted Critical
Publication of CN100488562C publication Critical patent/CN100488562C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Urology & Nephrology (AREA)
  • Oncology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明涉及新的凝血因子VII(FVII)或凝血因子VIIa(FVIIa)的多肽偶联物，其制备及在其治疗方面的用途，特别是对多种凝血相关疾病治疗的用途。这些新的多肽偶联物包含至少一个共价偶联于多肽上的非多肽组分，其中该多肽的氨基酸序列与野生型FVII或FVIIa不同在于其引入或去除了至少一个包含所述非多肽组分连接基团的氨基酸残基。本发明的偶联物与商品化的rFVIIa相比有一种或多种改进的特性，包括功能性体内半寿期延长和/或血浆半寿期延长，和/或生物利用率增加和/或对蛋白酶解的敏感性降低。The present invention relates to novel polypeptide conjugates of coagulation factor VII (FVII) or coagulation factor VIIa (FVIIa), their preparation, and their use in the treatment of various coagulation-related diseases. These novel polypeptide conjugates comprise at least one non-polypeptide component covalently coupled to a polypeptide, wherein the amino acid sequence of the polypeptide differs from that of wild-type FVII or FVIIa by the introduction or removal of at least one amino acid residue comprising a linking group for the non-polypeptide component. The conjugates of the present invention have one or more improved properties compared to commercial rFVIIa, including an increased functional in vivo half-life and/or an increased plasma half-life, and/or an increased bioavailability and/or a reduced susceptibility to proteolysis.

Description

Labile factor II or V II a sample molecule
Invention field
The present invention relates to new proconvertin (FVII) or proconvertin a (FVIIa) polypeptide conjugate, its preparation reaches the purposes in treatment, particularly treats the purposes of multiple blood coagulation relevant disease.
Background of invention
The process that causes fibrin clot to form gradually that blood coagulation is made up of complex interactions between the multiple blood constitutent (or factor).The blood constitutent that common participation is called as blood coagulation " cascade reaction " is protoenzyme or proenzyme,, does not have the albumen of enzymatic activity that is, and the effect of activator can make it change activity form into.FVII is exactly a kind of in these thrombins.
FVII is a kind of vitamin k-dependent plasma protein, and is synthetic in liver, and is that the strand glycoprotein form of 53kDa is secreted into (Broze ﹠amp in the blood with the molecular weight; Majerus, J.Bio1.Chem 1980; 255:1242-1247).The FVII proenzyme, is produced the two strands that is connected by a disulfide bond, thereby changes activity form (FVIIa) into by protease hydrolysis at R152-I153 place, single site.The complex of FVIIa and tissue factor (FVIIa complex) can all change plasma thromboplastin component and Stuart factor into its activity form, and ensuing reaction causes thrombin generation and fibrin formation (Osterud ﹠amp fast; Rapaport, Proc Natl Acad Sci USA 1977; 74:5260-5264).
FVII experiences post translational modification, comprises the carboxylated of vitamin K dependent, causes at 10 gamma-carboxyl glutamate residues of this molecule N-terminal district generation.Therefore, among the SEQ ID NO:1 the 6th, 7,14,16,19,20,25,26,29 and 35 residues are for the active important gamma-carboxyl glutamate residue of FVII in the Gla domain.Other post translational modifications are included in the N-glycosylation site of two natural generations of the 145th and 322, and are connected saccharic composition the 52nd respectively with the O-glycosylation site place of two natural generations of 60.
The gene of coding people FVII (hFVII) is located in chromosomal q34-qter9 (de Grouchy etc., Hum Genet 1984 No. 13; 66:230-233).It comprises 9 exons, and length is 12.8Kb (O ' Hara etc., Proc Natl Acad Sci USA 1987; 84:5158-5162).The structural similarity of the gene organization of FVII and protein structure and the former blood coagulating protein of other vitamin k-dependent, exons 1 a and 1b coded signal sequence; Exon 2 coded polypeptide and Gla domain; The hydrophobic region of one section weak point of exon 3 coding; Exon 4 and 5 coding schedule skin growth factor spline structure territories; And the catalyst structure domain of exon 6 to 8 encoding serine protease (YOshitake etc., Biochemistry 1985; 24:3736-3750).
Utilize X ray crystal imaging method (Banner etc., Nature, 1996; 380:41 and Zhang etc., J.Mol.Biol, 1999; 285:2089), hFVIIa (Pike etc., PNAS.U.S.A., 1999 have been reported; 96:8925-30 and Kemball-Cook etc., J.Struct.Biol, 1999; 127-213-223), the complex of hFVIIa and soluble tissue factor, and experiment three dimensional structure (Muranyi etc., Biochemistry, 1998 of the small fragment of hFVII; 37:10605 and Kao etc., Biochemistry, 1999; 38:7097).
About less relatively (the Dickinson ﹠amp of the report of FVII protein engineering mutant; Ruf, J BioChem, 1997; 272:19875-19879, Kemball-Cook etc., J Biol Chem, 1998; 273:8516-8521, Bharadwaj etc., J Biol Chem, 1996; 271:30685-30691, Ruf etc., Biochemsitry, 1999; 38:1957-1966).
Reported the expression of FVII in BHK or other mammalian cells (WO92/15686, WO91/11514, WO88/10295), and FVII and the coexpression (WO00/28065) of kex2 endo protease in eukaryotic cell.
The people recombinate FVIIa the commercialization preparation with
Figure C01804864D0006090152QIETU
Title sell.
Figure C01804864D0006090210QIETU
Indicate the bleeding episodes that is used for the treatment of hemophilia A or B patient. Be the unique effective and reliable rFVIIa of commercially available treated bleeding episodes.
Reported a kind of inactive form of FVII among the WO91/1154, wherein 152 arginine and/or 153 isoleucine are modified.These aminoacid are positioned at the activation site.WO96/12800 has described the inactivation of the FVIIa that a kind of serpin causes; Petersen etc. have illustrated that FVIIa is in inactivation (Eur J Biochem, 1999 that the alpha amino acid group carbamylation of I153 causes; 261:124-129).This inactivation form can with wild type FVII or FVIIa competition to tissue factor combine and anticoagulant activity.This points out the inactivation form of this FVIIa to can be used for treating the patient who very easily produces blood coagulation, as suffering from the patient of sepsis, its be easy to show effect myocardial infarction or thrombosis apoplexy.
At " Summary Basis for Approval for " reported that the circulation half life of rFVIIa is 2.3 hours in (FDA reference number 96-0597).High relatively dosage and continually administration be essential for the curative effect and the preventive effect that reach and keep expectation.Therefore, be difficult to obtain enough dosage regulation and control, and intravenously administrable has caused restriction for patient's life style continually.
Another problem of rFVIIa treatment at present is the relatively instability of this molecule for proteolysis.Opposite with the lyophilized powder product, proteolysis is a major obstacle that obtains solution type preparation.The benefit that obtains stable solution type preparation is to make things convenient for the patient to operate, and, in case of emergency, be convenient to work quickly, and this might be a kind of lifesaving.In WO88/10295, announced and attempted by major protein enzymolysis site being carried out the hydrolysis of direct mutagenesis prevention protease.
Molecule with longer circulation half life will reduce essential administration number of times.In view of existing FVIIa needs often injection, and probably when obtaining better therapeutic FVIIa level, heighten the effect of a treatment, clearly need to improve FVIIa or FVIIa sample molecule.
A kind of method that prolongs proteic circulation half life is to guarantee that this proteic renal clearance reduces.This can be by realizing this albumen and a kind of chemical constituent coupling this chemical constituent can be given the renal clearance that this albumen reduces.
In addition, chemical constituent and this proteic connection, or to being exposed to the amino acid whose replacement of proteolysis effect, avoid this proteic degraded thereby can effectively stop protease to contact.Polyethylene Glycol (PEG) is exactly a kind of such chemical constituent, can be used for preparing the human cytokines product.
WO98/32466 prompting, FVII is the same with other many albumen, can be by PEGization, but do not provide further information to this document.
The invention summary
The application discloses improved FVII and FVIIa molecule, particularly Chong Zu hFVII and hFVIIa molecule, and it provides one or more above-mentioned desired benefits.So, conjugate of the present invention and existing commercial rFVIIa relatively provide one or more improved characteristics, comprise having prolonged in the function gonosome half life and/or having prolonged the blood plasma half life, and/or increased bioavailability and/or reduced sensitivity proteolysis.Therefore, use conjugate of the present invention to treat can to obtain the multiple advantage that is better than existing rFVIIa complex, longer as injection interval.
Accordingly, the present invention relates to comprise the conjugate of at least one covalently bound non-polypeptide fractions to polypeptide on the one hand, wherein this amino acid sequence of polypeptide sequence of being different from wild type FVII shown in the SEQ ID NO:1 or FVIIa is that it introduces or remove an amino acid residue that comprises described polypeptide linking group at least.
Another aspect of the present invention relates to a peptide species, and wherein this amino acid sequence of polypeptide aminoacid sequence of being different from wild type FVII among the SEQID NO:1 or hFVIIa is that it introduces or remove an amino acid residue that comprises described polypeptide linking group at least.This new FVII polypeptide be considered to can be used for the treatment, the diagnosis and other purposes, but interested especially be as the preparation conjugate of the present invention intermediate product.
On the other hand, the present invention relates to: the nucleotide sequence of code book invention polypeptide or conjugate polypeptide portion of the present invention; The expression vector that comprises nucleotide sequence of the present invention; The host cell that comprises nucleotide sequence of the present invention or expression vector of the present invention.
The invention further relates to the pharmaceutical composition that comprises conjugate of the present invention and the method for preparation and this conjugate of use.
Detailed Description Of The Invention
Definition
In the application and full text of the present invention, use following definition:
Term " conjugate " (or interchangeable be " link coupled polypeptide ") expression is by one or more polypeptide and one or more non-polypeptide fractions (as polymer molecule, lipophilic compound, saccharic composition and organic derivating agent) covalently bound heterozygosis that forms (the compound or chimeric meaning) molecule.Preferably, conjugate is soluble under relevant concentration and condition, and is promptly solvable in physiological fluid such as blood.The embodiment of coupling polypeptide of the present invention comprises the polypeptide of glycosylated and/or PEGization.
This polypeptide and non-polypeptide fractions or directly covalently bound each other represented in term " covalently bound ", perhaps by one or more components that interleave, as bridge, introns or coupling part, covalently bound indirectly.
Term " non-coupling polypeptide " is the polypeptide portion of conjugate.
Term used herein " non-polypeptide fractions " expression can with the link coupled molecule of the linking group of polypeptide of the present invention.The preferred embodiment of this quasi-molecule comprises polymer molecule, saccharic composition, lipophilic compound or organic derivating agent.When being used for conjugate of the present invention in this article, be interpreted as non-polypeptide fractions is connected to conjugate by the polypeptide linking group polypeptide portion.As above explanation, non-polypeptide fractions may directly covalently be connected to linking group, or by one or more components that interleave, as bridge, introns or coupling part, covalently bound indirectly to linking group.
The term " polymer " molecule " be defined as by the covalently bound molecule that forms of two or more monomers, none is amino acid residue for wherein said monomer, removing non-polymer is that human albumin or another kind enrich plasma protein.Term " polymer " " can with the term " polymer " molecule " exchange.The carbohydrate molecule that connects through external glycosylation contained in this term, and described external glycosylation is the external synthetic property glycosylation of carrying out, and it is usually directed to carbohydrate molecule and the polypeptide linking group is covalently bound, the optional cross-linking agent that uses.
The carbohydrate molecule that glycosylation such as N-or O-glycosylation (hereinafter being further described) connect in the body is called " saccharic composition " in this article.The number of non-polypeptide fractions such as polymer molecule or saccharic composition, the one or more non-peptide moiety in the conjugate in " the non-polypeptide fractions " of other parts indication contained or of the present invention expression conjugate is as polymer molecule or saccharic composition in indicating conjugate.
Term " linking group " expression polypeptide can with non-polypeptide fractions such as polymer molecule, saccharic composition, lipophilic compound or the link coupled functional group of organic derivating agent, particularly its amino acid residue or carbohydrate ingredient.Useful linking group and the non-polypeptide fractions that matches thereof are illustrated in figure below.
Linking group Aminoacid Non-polypeptide fractions example Coupling method/activatory PEG Reference
-NH 2 The N-end, lysine Polymer as PEG, has amide or imine group mPEG-SP A TresylatedmPEG Shearwater Inc.Delgado etc., critical reviews in Therapeutic Drug Carrier Systems 9 (3,4): 249-304 (1992)
-COOH The C-end, aspartic acid, glutamic acid Polymer as PEG, has ester or amide group carbohydrate ingredient The external coupling of mPEG-Hz Shearwater Inc.
-SH Cysteine Polymer as PEG, has disulfide bond, maleimide or vinyl sulfone(Remzaol group carbohydrate ingredient The external coupling of PEG-vinyl sulfone(Remzaol PEG-maleimide Shearwater Inc.Delgado etc., critical reviews in Therapeutic Drug Carrier Systems 9 (3,4): 249-304 (1992)
-OH Serine, threonine, lysine, OH- Saccharic composition has the PEG of ester, ether, carbamate, carbonate group The glycosylation that O-connects in the body
-CONH 2 Agedoite is as part N-glycosylation site The saccharic composition polymer is as PEG N-glycosylation in the body
Aromatic residue Phenylalanine, tyrosine, tryptophan Carbohydrate ingredient External coupling
-CONH 2 Glutamine Carbohydrate ingredient External coupling Yan & Wold，Biochemistry， 1984，Ju131：23(16)：3759-65
Aldehyde ketone The oligosaccharide of oxidation Polymer, as PEG, the PEG-hydrazides PEGization Andresz etc., 1978, Makromol.Chem.179:301, WO92/16555, WO00/23114
Guanidine Arginine Carbohydrate ingredient External coupling Lundblad & Noyes， Chemical Reagents for Protein Modification，CRCPress Inc.，Florida USA
Imidazole ring Histidine Carbohydrate ingredient External coupling Same guanidine
For N-glycosylation in the body, term " linking group " is used to represent to constitute the amino acid residue of N-glycosylation site in unconventional mode, and (sequence is N-X-S/T/C, wherein X is any amino acid residue except that proline, N is that agedoite and S/T/C are serine, threonine or cysteine, preferably serine or threonine and most preferably threonine).Although the asparagine residue of N-glycosylation site is the residue that is connected with saccharic composition during glycosylation, this connection can not be finished, unless there is other amino acid residue in this N-glycosylation site.
Therefore, when non-polypeptide fractions is a saccharic composition, and coupling is when realizing by the N-glycosylation, one or more amino acid residues that the term " amino acid residue that contains non-polypeptide fractions linking group " that changes coupling with the desired polypeptides aminoacid sequence is interpreted as constituting the N-glycosylation site are changed, and shifting gears is that functional N-glycosylation site is introduced into aminoacid sequence or removes from described sequence.
In this application, the name of aminoacid name and atom (as, CA, CB, CD, CG, SG, NZ, N, O, C etc.) by (www.pdb.org) the defined use of Protein DataBank (PDB), this definition is based on the IUPAC nomenclature (symbol of IUPAC nomenclature and aminoacid and peptide (residue name, atom name etc.), Eur.J.Biochem., 138,9-37 (1984) with and the Eur.J.Biochem. that corrects errors in printing, 152,1 (1985)).
Term " amino acid residue " expression is included in down the amino acid residue in the group: alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), agedoite (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y) residue.
Be used for identifying that the term of amino acid position illustrates as follows: G124 represents that the 124th of aminoacid sequence shown in the SEQ ID NO.1 occupied by glycine residue, and G124R represents that the 124th glycine residue replaced by arginine residues.Alternative replacement can "/" be represented, represents that as K32D/E the 32nd lysine is replaced by aspartic acid or glutamic acid.Polysubstituted with "+" expression, represent that as K143N+X145S/T the 143rd lysine residue replaced by asparagine residue, and the 145th asparagine residue is replaced by serine residue or threonine residues.The insertion of additional amino acid as insert an alanine residue after G124, is expressed as G124GA.The disappearance of amino acid residue is represented with asterisk.For example, the disappearance of the 124th glycine is expressed as G124 *Except as otherwise noted, herein the sequence number of amino acid residue corresponding to the aminoacid sequence of wild type FVII/FVIIa shown in the SEQ ID NO:1.
The term that used herein and specific sudden change is relevant " is different from " and is meant that removing the specific unusual permission of amino acid difference exists other difference.For example, comprise the amino acid residue of non-polypeptide fractions linking group except removing and/or introducing, FVII or FVIIa polypeptide may also comprise introducing and/or other the irrelevant replacements of removal with these amino acid residues.Therefore, (these amino acid change purposes are except the amino acid change announced herein, remove and/or introduce the linking group of non-polypeptide fractions) outside, can understand amino acid sequence of polypeptide of the present invention, can comprise other changes if desired, these change not necessarily must with the introducing of connection site or remove relevant, i.e. other replacement, insertion or disappearance.For example, these changes may comprise one or more amino acid residues of removing N-and/or C-terminal, or introduce one or more extra amino acid residues at N-and/or C-terminal, as adding a methionine residues at N-terminal, and " conservative amino acid replacement ", that is, replacement is to have the aminoacid of identical characteristics, for example, p1 amino acid, acidic amino acid, polar amino acid, basic amino acid replaces between hydrophobic amino acid and the aromatic amino acid.
Preferred replacement is selected from the listed conservative replacement group of following table especially among the present invention.
1 Alanine (A) glycine (G) serine (S) threonine (T)
2 Aspartic acid (D) glutamic acid (E)
3 Agedoite (N) glutamine (Q)
4 Arginine (R) histidine (H) lysine (K)
5 Isoleucine (I) leucine (L) methionine (M) a word used in person's names propylhomoserin (V)
6 Phenylalanine (F) tyrosine (Y) tryptophan (W)
Term " sudden change " and " replacement " are used interchangeably at this.
The continuous fragment of the two or more nucleic acid molecules of term " nucleotide sequence " expression.Nucleotide sequence can be genome, cDNA, RNA, semi-synthetic or synthetic source or its combination in any.
Term " polymerase chain reaction " or " PCR " are often referred to a kind of method that is used for the required nucleotide sequence of amplification in vitro, for example, press the method that United States Patent (USP) 4683195 is described.In general, PCR method relates to use and can the primer extension synthetic reaction be circulated repeatedly with the oligonucleotide primers of the preferential hybridization of template nucleic acid
" cell ", " host cell ", " cell line " and " cultured cell " are used interchangeably in this article and these terms all should be understood to comprise the cell growth or cultivate the offspring who is produced." conversion " and " transfection " is used interchangeably, and refers to DNA is introduced the process of cell.
" can be operatively connected " and refer to that two or more nucleotide sequences are covalently bound in the conformation that is relative to each other by modes such as enzymatic connections, make sequence exercise normal function.For example, if the nucleotide sequence of coding presequence or secretion targeting sequencing be expressed as participate in polypeptide excretory before albumen, then listed by the nucleotides sequence that operability is connected to this polypeptide; If promoter or enhancer can influence transcribing of this sequence, then be connected with the coded sequence operability; If ribosomal binding site is in the position that promotes translation then can be operatively connected with coded sequence.Usually, " can be operatively connected " means connected nucleotide sequence is adjacency, and the secretion boot section situation under, be adjacency and also under read state.Being connected easily restriction site finishes.The recombinant DNA method of synthetic oligonucleotide adapter or joint and standard is used in if there is no this type of site so.
Term " introducing " mainly refers to comprise the introducing of the amino acid residue of non-polypeptide fractions linking group, particularly by replacing existing amino acid residue, perhaps by inserting other amino acid residue.
Term " removal " mainly refers to comprise the removal of the amino acid residue of non-polypeptide fractions linking group, particularly by this amino acid residue is removed with the mode that other amino acid residue replaces, perhaps makes the amino acid residue disappearance (not replacing) of pre-removal.
Term " FVIIa " or " FVII " polypeptide refer to the FVII molecule of single stranded form.
Term " FVIIa " or " FVIIa polypeptide " refer to the FVIIa molecule of activatory double chain form, and the peptide bond between wherein said single stranded form R152 and the I153 is sheared.When the aminoacid sequence of SEQ ID NO:1 is used to the aminoacid sequence of FVIIa is described herein, should understand wherein the amino acid residue that a chain comprises the 1-152 position, another chain comprises the amino acid residue of 153-406 position.
Term " rFVII " and " rFVIIa " refer to FVII and the FVIIa molecule that recombinant technique produces respectively.
Term " hFVII " and " hFVIIa " refer to wild type people FVII and FVIIa respectively.
Term " catalytic site " is used in reference to the S344 by the FVII polypeptide, the catalysis triplet that D242 and H193 form.
Term " active FVIIa ", " active FVIIa polypeptide ", " active FVIIa conjugate " or " active conjugate " are used in reference to FVIIa polypeptide or the conjugate with wild type hFVIIa catalytic activity of at least 10%.In the literary composition used catalytic activity can be suitably by at the method chapters and sections that detect catalytic activity or detect in the method chapters and sections of low-level catalytic activity illustrated method and determine (material and method one joint in referring to literary composition).When analyzing with the above, described active conjugate preferably has at least 15% of wild type hFVIIa catalytic activity, and for example at least 20%, as at least 25%, more preferably at least 30%, as at least 40%, most preferably at least 50%, as at least 60%.
Preferably, active conjugate can bind tissue factor, and further activates plasma coagulation factors X and/or IX.Therefore, in a preferred embodiment, active FVIIa polypeptide or its conjugate are compared with wild type FVIIa, have at least 25% blood coagulation activity, have at least 50% blood coagulation activity as comparing, have at least 75% blood coagulation activity as comparing with wild type FVII with wild type FVIIa.Therefore, the blood coagulation activity of active FVIIa polypeptide or its conjugate is compared with wild type FVIIa, preferably in the scope of 25-200%.Particularly, the blood coagulation activity of preferred FVIIa polypeptide or its conjugate is compared in the scope of 30-150% with wild type FVIIa, compares in the scope of 30-100% with wild type FVIIa as blood coagulation activity.Blood coagulation activity can detect by any method known in the art, as further discussing in material and method one joint.Yet the preferred especially method according to explanation in " detecting the method for blood coagulation activity " chapters and sections (referring to material and method part) is determined blood coagulation activity.
With the term " immunogenicity " of given material coupling but be intended to represent the ability of this material induction of immunity system response.Immunoreation can be cell or antibody-mediated reaction (to immunogenicity further define referring to, as, Roitt:Essential Immunnology (8th Edition, Blackwell)).In general, antibody response decline prompting immunogenicity descends.Immunogenicity can be measured by any known method in this area, as method in the body or in vitro method.
Term " non-activity FVIIa ", " non-activity FVIIa polypeptide ", " the FVIIa conjugate of non-activity " or " non-activity conjugate " are used in reference to 10% FVIIa polypeptide or the conjugate that activity is lower than wild type hFVIIa catalytic activity.In the literary composition used catalytic activity can be suitably in method chapters and sections by detecting catalytic activity or the method chapters and sections that detect low-level catalytic activity illustrated method determine (referring to material in the literary composition and method chapters and sections).When adopting the above to analyze, the catalytic activity of non-activity conjugate preferably is lower than 8% of wild type hFVIIa catalytic activity, as 6%, for example is lower than 5%, more preferably less than 4%, as is lower than 3%, most preferably is lower than 2%, for example is lower than 1%.
Usually, the non-activity conjugate is compared with wild type hFVIIa, and external or body intravascular coagulation is active significantly to be reduced.The FVII of this non-activity or FVIIa polypeptide or conjugate may with wild type FVII or FVIIa competition bind tissue factor, thereby anticoagulant activity.Preferred this non-activity FVII or FVIIa polypeptide or conjugate are compared with wild type hFVII or hFVIIa, and blood coagulation activity is lower than 1% of wild type.More preferably this non-activity FVII or FVIIa polypeptide or conjugate are compared with wild type hFVII or hFVIIa, and blood coagulation activity is lower than 0.05% of wild type.Most preferably this non-activity FVII or FVIIa polypeptide or conjugate are compared with wild type hFVII or hFVIIa, and blood coagulation activity is lower than 0.01% of wild type.Blood coagulation activity can detect by any method known in the art in the same manner as described above, as further discussing in material and method.Yet the preferred especially method according to explanation in " detecting the method for blood coagulation activity " chapters and sections (referring to material and method part) is determined blood coagulation activity.
Term " function gonosome in half life " uses its common implication, promptly polypeptide or conjugate in vivo/still had for 50% bioactive time in the target organ, perhaps the activity of polypeptide or conjugate is time of 50% of initial activity.As the another kind of method of half life in the measurement function gonosome, can measure " serum half life ", that is, and 50% polypeptide or conjugate molecule before being eliminated in blood plasma or blood flow the circulation time.The mensuration of serum half life usually than half life in the measurement function gonosome size of simple and serum half life usually well in the functions gonosome half life size.Other replaceable term of serum half life comprises " blood plasma half life ", " circulation half life ", " serum clearance rate ", " plasma clearance " and " removing half life ".This polypeptide or conjugate be by one or more effect in reticuloendothelial system (RES), kidney, spleen or the liver, by tissue factor, SEC receptor or other receptor-mediated scavenging actions, or removes by special or non-specific proteolysis.Usually remove and depend on the carbohydrate chain of size (for the cutoff value of glomerular filtration), electric charge, connection, and whether have this proteic cell receptor.The function that keeps is selected from procoagulant, proteolysis or receptor-binding activity usually.Half life and serum half life, can be measured by any suitable method as known in the art in the function gonosome, and assay method material and method joint hereinafter is discussed further.
The term " increase " that relates to half life in the function gonosome or blood plasma half life is used to represent the mensuration of corresponding half life under comparable conditions of conjugate or polypeptide, with respect to the reference molecule as not link coupled rFVIIa (as
Figure C01804864D00141
) half life be significantly to increase statistically.For example, corresponding half life, may increase at least about 25%, as at least about 50%, for example increases at least about 100%, 150% 200%, 250%, 300%, 500% or 100%.
Term " kidney removing " uses its common implication, promptly occurs in the removing of kidney, for example, finishes by glomerular filtration, tubular excretion or the degraded in renal tubular cell.Kidney is removed the physical characteristic that depends on conjugate, comprises size (diameter) fluid volume, symmetry, shape/rigidity and electric charge.As a rule, the molecular weight that is approximately 67kDa is considered to the cutoff value that kidney is removed.Kidney is removed and can be set up by any suitable assay method, as assay method in the body of having set up.Usually, kidney remove by with labelling (as, radiolabeled or fluorescently-labeled) the polypeptide conjugate gives the patient and the label activity measured in patient's urine of collecting is measured.The decline that kidney is removed through under the comparable conditions with corresponding with reference to polypeptide, as corresponding non-coupling polypeptide, non-link coupled corresponding wild type peptide, or other coupling polypeptide (as the coupling polypeptide that has nothing to do with the present invention) compare and determine.Preferably, the renal clearance of conjugate is than reducing at least 50% with reference to polypeptide accordingly, and preferred reduction at least 75% most preferably reduces at least 90%.
Conjugate performance of the present invention is very important to the character that proteolysis sensitivity reduces; The compositions that comprises hydrolyzate is not hydrolyzed with those conjugates or the compositions that only has fraction to be hydrolyzed is compared, and has littler specificity usually.In addition, non-physiology catabolite may excite patient's immune system in the administration composition.
Term " to the sensitivity of proteolysis reduction " mainly refers to the detection under comparable conditions, and this conjugate is compared with non-link coupled wild type FVIIa, to the sensitivity reduction of proteolysis.Preferably, proteolysis has reduced at least 10%, as at least 25% (for example having reduced 10%-25%), more preferably reduced at least 35%, (for example reduced 10%-50%,, more preferably reduced by 60% as having reduced by 50% at least as 25%-50%, as having reduced by 75% at least, or even reduced at least 90%.More preferably, proteolysis has reduced by 100%.Therefore, it is little that preferred conjugate of the present invention and wild type FVIIa compare the proteolysis degree, promptly compare with non-link coupled wild type FVIIa, the proteolysis of conjugate of the present invention has preferably reduced 10%-100%, as reduced 25%-100%, more preferably reduce 50%-100%, most preferably reduced 75%-100%.
The inventor has set up a kind of suitable preliminary external detection method, and it can be used for assessing this conjugate whether the sensitivity of proteolysis is reduced (minimizing of oneself protein enzymolysis).Therefore, in the preferred embodiment of the invention, the method detection of explanation in method in method, " the detection catalytic activity " of explanation in " detection that proteolysis sensitivity is reduced " joint save or " detecting low-level catalytic activity method " joint (referring to this paper material and method joint), the sensitivity that conjugate of the present invention and wild type FVIIa compare proteolysis reduces.
Term " parental generation FVII " or " parental generation polypeptide " are meant among the present invention adorned molecule.Typical parental generation FVII be hFVII or hFVIIa (comprise rfVIIa (
Figure C01804864D0015091450QIETU
)), it has aminoacid sequence shown in the SEQ IDNO:1.
" variant " is a peptide species, and itself and parental generation polypeptide have the difference of one or more amino acid residues, has 1,2,3,4,5,6,7,8,9,10,11,12 usually, the difference of 13,14 or 15 amino acid residues.
Conjugate of the present invention
Conjugate of the present invention is that the overall New Policy of exploitation FVII or FVIIa improvement molecule produces.More specifically, by removing and/or introduce the amino acid residue of the linking group that contains described non-polypeptide fractions, changing polypeptide specifically is easier to and selected non-polypeptide fractions coupling this molecule, make coupling pattern optimization (as, guarantee non-polypeptide fractions with the optimal dose best distribution at FVII or FVIIa molecular surface and guarantee only to exist link coupled linking group in the molecule), thereby obtain new coupling molecule, it is compared with the FVIIa molecule with existing FVII, have or do not have the activity of FVII, have one or more improved characteristics in addition.For example, when the amino acid residue sum of the linking group that contains selected non-polypeptide fractions was increased to or is reduced to an optimum level, the change of the molecular shape that the renal clearance of this conjugate causes because of coupling usually, size and/or electric charge significantly reduced.
In the preferred embodiment of the invention, the more than one amino acid residue of FVII or FVIIa polypeptide is changed, and for example described change comprises the amino acid residue of removing and introducing the linking group that comprises selected non-polypeptide.Except removing and/or introduce the amino acid residue, this polypeptide may comprise other with the removal of the amino acid residue of non-polypeptide fractions linking group with introduce replacement or the glycosylation that has nothing to do.This polypeptide also may be connected with serpin, to suppress the catalytic site of this polypeptide.
No matter the amino acid residue that comprises non-polypeptide fractions linking group is to be removed or to introduce, and it selects the character based on selected non-polypeptide fractions, and, in most cases, based on coupling between polypeptide and the non-polypeptide fractions all method.For example, when non-polypeptide fractions is a polymer molecule, as Polyethylene Glycol or deutero-minute period of the day from 11 p.m. to 1 a.m of polyalkylene oxide, the amino acid residue that comprises linking group can be selected from: lysine, cysteine, aspartic acid, glutamic acid, histidine, or tyrosine, preferred cysteine and lysine, preferred especially lysine.
Non-polypeptide fractions linking group is introduced into when being removed in FVII of the present invention or the FVIIa polypeptide or from described polypeptide, carry out the modified polypeptides position and be preferably placed at the surface of this polypeptide, be more preferably by there being 25% above side chain to be exposed to the position that those amino acid residues in the solvent occupy, preferred described aminoacid is exposed in the solvent above 50% side chain.Identified these positions through the analysis to people FVII or FVIIa molecule three dimensional structure, described method is seen this paper materials and methods one joint.In addition, this optimum seeking site is to be positioned at outside the tissue factor binding site district in the FVII molecule and/or the part outside the avtive spot district.These zones are discerned in material and the method hereinafter.Yet what be worth emphasizing is, it is favourable (obtaining the conjugate of non-activity as expectation) under some situation in these zones or suddenling change near it.For example, think one or more linking groups of non-polypeptide fractions, as the linking group of N-glycosylation site in the body, it is favourable being inserted on the ridge of the activity site district of FVII molecule or activity site engagement groove.Defined the ridge of this avtive spot district and avtive spot engagement groove hereinafter in the materials and methods, it is made of following residue:
I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379, V380, Q382, Y383, W386, L387, L400 and F405 (activity site district); And N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370 (ridge of activity site engagement groove)
In order to determine the best distribution of linking group, calculate the distance between the amino acid residue on this polypeptide surface based on the three dimensional structure of FVII or FVIIa molecule.More specifically, to the distance between the CB of the amino acid residue that comprises this linking group, or a kind of amino acid whose functional group (NZ of lysine, the CG of aspartic acid, the CD of glutamic acid, the SG of cysteine) and the distance that comprises between the CB of another kind of amino acid residue of linking group determine.For glycine, use CA but not CB.In the FVII or FVIIa polypeptide portion of conjugate of the present invention, any described distance is preferably greater than
Figure C01804864D0017091611QIETU
Be preferably greater than 10 especially, to avoid or to reduce the allos coupling.
When removing linking group, the corresponding amino acid residue that comprises this group and occupy the above position is preferably with a kind of different amino acid residues replacement, and this amino acid residue does not comprise the linking group of described non-polypeptide fractions.Usually, the amino acid residue that will remove is to produce unfavorable link coupled amino acid residue, as is positioned at polypeptide functional site or near the amino acid residue it (because the coupling in these sites may cause the conjugate inactivation that produced or the activity of FVII or FVIIa to reduce because of destroying receptor identification).Term " functional site " is meant for the function of FVII or FVIIa or acts on requisite or relevant amino acid residue herein.This amino acid residue is the part of described functional site.This functional site can determine by means known in the art, and preferably (referring to Banner etc., Nature 1996 by the structural analysis of FVIIa-tissue factor complex is discerned; 380:41-46).
When introducing linking group, the amino acid residue that comprises this group is introduced into this position, and preferably the amino acid residue that takes up position by replacement is realized.
The definite quantity that exists in FVII or the FVIIa polypeptide and can be used for link coupled linking group depends on the effect that expectation reaches by coupling.The effect that obtains depends on, for example link coupled nature and extent (determining of non-polypeptide fractions for example, expectation or may with the number of the link coupled non-polypeptide of polypeptide, the position that coupling takes place etc. maybe should be avoided in the position that coupling should take place).
Half life, depend on the molecular weight of conjugate in the function gonosome, and therefore the number that prolongs required linking group of half life depends on the molecular weight of described non-polypeptide fractions.In one embodiment, when by Laemmli, U.K., when the SDS-PAGE among Nature Vol 227 (1970) .P680-85 measured, conjugate of the present invention had 67kDa at least, particularly the molecular weight of 70kDa at least.FVII has the molecular weight of about 53kDa, therefore, for obtaining desired effect, needs to add in addition about 10-20kDa.This can for example pass through, and the PEG molecule of 2 to 4 10kDa of coupling or other method described herein realize.
Cause too many destruction for fear of the 26S Proteasome Structure and Function to the parental generation molecule, the polypeptide portion of conjugate has and the homogeneity of aminoacid sequence more than 90% shown in the SEQ ID NO:1 usually, and is preferred more than 95%, as more than 96%.Concrete, the conjugate polypeptide portion has and the homogeneity of aminoacid sequence more than 97% shown in the SEQ ID NO:1 usually, as more than 98%, more than 99%, more than 99.25%, more than 99.25% or more than 99.5%.
Amino acid sequence homology/homogeneity can be used expediently as ClustalW program (version 1.8, in June, 1999), use default parameter to determine (Thompson etc. through sequence alignment, 1994, ClustalW:Improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research, 22:4673-4680), or by using GENEDOC version 2 .5 (Nicholas, K.B., Nicholas H.B.Jr., and Deerfield, D.W.II.1997 GeneDoc:Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas, K.B.and Nicholas H.B.Jr.1997 GeneDoc:Analysis and Visualizationof Genetic Variation) from the information bank edition 4 .0 of PEAM family (http://pfam.wustl.edu/) (Nucleic Acids Res.1999 Jan 1; 27 (1): determine 260-2).
Change a kind of saying, the sum (comparing with SEQ ID NO:1 aminoacid sequence) according to the present invention with the amino acid residue that changes is no more than 15 usually.Preferably, the FVII of conjugate of the present invention or FVIIa polypeptide portion or polypeptide of the present invention comprise an aminoacid sequence, aminoacid sequence has 1-15 amino acid residue shown in itself and the SEQ ID NO:1, the difference of 1-10 or 2-10 amino acid residue normally, for example 1-8 or 2-8 amino acid residue, the difference of or 4-6 amino acid residue individual as 3-7.Therefore, usually the polypeptide portion or the polypeptide of the present invention of conjugate comprise and the different aminoacid sequence of aminoacid sequence shown in the SEQ ID NO:1, its difference is at most 15 amino acid residues (as 15 amino acid residues), 14 amino acid residues (as 14 amino acid residues) at the most, 13 amino acid residues (as 13 amino acid residues) at the most, 12 amino acid residues (as 12 amino acid residues) at the most, 11 amino acid residues (as 11 amino acid residues) at the most, 10 amino acid residues (as 10 amino acid residues) at the most, 9 amino acid residues (as 9 amino acid residues) at the most, 8 amino acid residues (as 8 amino acid residues) at the most, 7 amino acid residues (as 7 amino acid residues) at the most, 6 amino acid residues (as 6 amino acid residues) at the most, 5 amino acid residues (as 5 amino acid residues) at the most, 4 amino acid residues (as 4 amino acid residues) at the most, 3 amino acid residues (as 3 amino acid residues), 2 amino acid residues (as 2 amino acid residues) at the most at the most.
Similarly, conjugate of the present invention comprises usually, as 1-15 non-polypeptide fractions, and common 1-10 non-polypeptide fractions or 2-10 non-polypeptide fractions, as 1-8 or 2-8 non-polypeptide fractions, as 1-6,1-4,3-7 or 4-6 non-polypeptide fractions.
Preferably, conjugate of the present invention and rFVIIa (as
Figure C01804864D0018091903QIETU
) compare, have one or multinomial following improved characteristic: half life, prolong in the function gonosome; The blood plasma half life, prolong, and renal clearance reduces and the sensitivity of proteolysis is reduced.
The proteolysis of knowing FVII/FVIIa from WO88/10295 occurs in a plurality of proteolysiss site in this molecule, promptly at K32, and K38, I42, Y44, K143, R290, R315, K341, R392, R396 and R402 (or are positioned at K32-D33, K38-L39, I42-S43, Y44-S45, K143-R144, R290-G291, R315-K316, K341-G342, R392-S393 is between R396-P397 and the R402-A403).Therefore, increasing half life in the function gonosome for example, increasing the blood plasma half life or reduce a preference policy to proteolysis sensitivity is to change the parental generation polypeptide near one or more sites in these proteolysis sites and/or its, and method therefor is to introduce non-polypeptide fractions on these sites or near it.Therefore, in a preferred embodiment of the present invention, on the above-mentioned one or more positions that identify, or with respect to the position of participating in proteolysis directly-4 ,-3 ,-2 ,-1,1,2,3,4, optimum position-2 ,-1,1,2, on position-1,1, introduced linking group.
More specifically, preferably N-glycosylation site (referring to following) in the body that glycosylation site, particularly non-natural produce in the body that non-natural is produced introduces being selected from following position: 28-48,139-147,286-294,311-319,338-345 and 388-406.Concrete, preferably with glycosylation site in the body,, introduce being selected from following position: 30-34,36-46,141-144,288-292,313-317,341-343,390-398 and 400-404 as N-glycosylation site in the body (referring to following).More preferably, with glycosylation site in the body, as N-glycosylation site in the body (referring to following), introducing is selected from following position: 31-33 (as 31,32 or 33), and 37-39 is (as 37,38 or 39), 41-46 is (as 41,42,43,45 or 46), 142-144 is (as 142,143 or 144), 289-291 (as 289,290 or 291), 314-316 is (as 314,315 or 316), 341-342 (as 341,342 or 343), 391-393 is (as 391,392 or 393), 395-397 (as 395,396 or 397) and 401-403 (as 401,402 or 403).
Conjugate of the present invention wherein is that non-polypeptide fractions is to have the molecule of lysine as linking group
In one embodiment of the invention, non-polypeptide fractions comprises lysine as linking group, promptly, conjugate of the present invention comprises at least one and the link coupled non-polypeptide fractions of polypeptid covalence, the aminoacid sequence that wherein said amino acid sequence of polypeptide is different from wild type FVII shown in the SEQ ID NO:1 or FVIIa is, its introducing or removed at least one amino acid residue.
FVII/FVIIa comprises 17 lysine residues.Three lysine residues (K18, K62 and K85) are positioned at the tissue factor binding structural domain, and two lysine residues (K197 and K341) are positioned at the avtive spot district.
Because the relative high-load of lysine residue in the parental generation polypeptide thinks that at least one lysine residue should preferably be removed, particularly remove, to avoid excessive coupling to non-polypeptide fractions by replacing lysine residue with non-lysine residue.
Therefore, in the embodiment, the aminoacid sequence of the FVII of conjugate or FVIIa polypeptide portion is with different shown in the SEQ ID NO:1, difference is, has preferably removed at least one lysine residue by replacement, as the 1-15 lysine residue, particularly 1-10,1-6 or 2-4 lysine residue.For example, preferably be selected from: K18, K32, K38, K62, K85, K109, K137, K143, K148, K157, K161, K197, K199, K316, K337, K341, K389 or its combination by replacing the lysine residue of removing.Concrete, preferably remove lysine residue, this residue is the ingredient in tissue factor binding site and/or avtive spot district, that is, and residue K18, K62, K85, K197, K341 or its compositions.This lysine residue can replace by all any other amino acid residues, but preferably by R, Q, N or H are more preferably replaced by R.
In another embodiment, the aminoacid sequence of the FVII of conjugate or FVIIa polypeptide portion is different with the sequence shown in the SEQID NO:1, difference is to have introduced at least one lysine residue, as the 1-15 lysine residue, 1-10 particularly, 1-6 or 2-4 lysine residue are preferably introduced by substituting.Can understand, introduce at least one lysine residue at parental generation molecule predetermined site, it is preferred making up with the removal of above-mentioned at least one lysine residue.Therefore, in embodiment preferred of the present invention, the aminoacid sequence of the FVII of conjugate or FVIIa polypeptide portion is different with the sequence shown in the SEQ ID NO:1, and difference is to have removed at least one lysine residue and has introduced at least one lysine residue.
The position that lysine residue can be introduced into includes, but are not limited near above-mentioned proteolysis site or its.Therefore, in the preferred embodiment, replacing non-lysine residue with lysine residue can be selected from: I42K, Y44K, L288K, D289K, R290K, G291K, A292K, T293K, Q313K, S314K, R315K, V317K, L390K, M391K, R392K, S393K, E394K, P395K, R396K, P397K, G398K, V399K, L400K, L401K, R402K, A403K, P404K, F405K with or its combination, be selected from especially: R290K, R315K, R392K, R396K, R402K or its combination.
According to the present invention this on the one hand, the non-polypeptide fractions of conjugate can be any molecule, when using given coupling method with lysine during as linking group, preferred non-polypeptide fractions is a polymer molecule.Described polymer molecule can be any molecule of mentioning in " with the polymer molecule coupling " joint, but is preferably selected from linearity or the PEG of branch or another polyalkylene oxide.The example of preferred polymer molecule is from Shearwater Polymers, the SS-PEG of Inc., NPC-PEG, aldehyde-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC; From Enzon, the SC-PEG of Inc.; The described tresylated mPEG of US5880255; Or oxygen carbonyl-oxygen-N-dicarboxyl acid imide-PEG (US5122614).
In general, during with the lysine residue coupling, the molecular weight of non-polypeptide fractions is about 5 to about 20kDa, for example from about 5 to about 10kDa, and 5kDa or about 10kDa according to appointment.
Should be appreciated that the described any amino acid change in this part, especially replace that can make up with any amino acid change, preferably the specific amino acids with the described specific replacement of other parts herein changes, comprise the introducing of glycosylation site and/or remove combination.
Conjugate of the present invention, wherein non-polypeptide fractions are to have the molecule of cysteine as linking group
In another embodiment of the invention, non-polypeptide fractions with cysteine as linking group, be that conjugate of the present invention comprises at least one and the link coupled non-polypeptide fractions of polypeptid covalence, wherein said amino acid sequence of polypeptide is different among the SEQ ID NO:1 wild type people FVII or the FVIIa part is, introduces or removed a cysteine residues at least.
FVII/FVIIa comprises 24 cysteine residues, and disulfide bond is set up between following cysteine residues: C17 and C22, C50 and C61, C55 and C70, C72 and C81, C91 and C102, C98 and C112, C114 and C127, C135 and C262, C159 and C164, C178 and C194, C310 and C329, and between C340 and the C368.
In another embodiment, different among the aminoacid sequence of FVII or FVIIa polypeptide portion and the SEQ IDNO:1, difference is to introduce at least one lysine residue, as 1-15 lysine residue, 1-10 particularly, a 1-6 or 2-4 lysine residue is preferably introduced by replacing.
The position that can introduce lysine residue includes, but are not limited near above-mentioned proteolysis site or its.
Therefore in one embodiment of the invention, preferably be selected from: I30C, K32C, D33C, A34C by replacing the lysine residue of introducing, T37C, K38C, W41C, Y44C, S45C, D46C, L141C, E142C, K143C, R144C, L288C, D289C, R290C, G291C, A292C, S314C, R315C, K316C, V317C, L390C, M391C, R392C, S393C, E394C, P395C, R396C, P397C, G398C, V399C, L401C, R402C, A403C, P404C or its combination are selected from: K32C especially, Y44C, K143C, R290C, R315C, K341C, R392C, R396C, R402C or its combination.
In the further embodiment of the present invention, cysteine residues is introduced into those threonine or the serine residue position occupied that is exposed to the surface among the wild type hFVII by the side chain with at least 25%.For example, at least one site of FVII or FVIIa polypeptide, preferably introduce cysteine residues by replacing, described site is selected from: S12, S23, S43, S45, S52, S53, S60, S67, T83, S103, T106, T108, S111, S119, S126, T128, T130, S147, T185, S214, S222, S232, T233, T238, T239, T255, T267, T293, T307, S320, T324, S333, S336, T370 and S393.More preferably at least one contains on the site of S residue and introduces cysteine residues among the hFVII, and described site is selected from: S12, S23, S43, S45, S52, S53, S60, S67, S103, S111, S119, S126, S147, S214, S222, S232, S320, S333, S336 and S393.
In the further embodiment of the present invention, cysteine residues is introduced among the wild type hFVII by having those threonine or the serine residue position occupied that 50% side chain is exposed to the surface at least.For example, preferred by replacing cysteine residues of introducing at least one position of FVII or FVIIa polypeptide, described position is selected from: S23, S43, S52, S53, S60, S67, T106, T108, S111, S119, S147, S214, T238, T267 and T293, more preferably described position is selected from: S23, S43, S52, S53, S60, S67, S111, S119, S147 and S214.
Further in the embodiment, cysteine residues is introduced at least one and is selected from above-mentioned arbitrary position and is not the position in avtive spot district.Preferred this position is occupied by T or S residue.For example, the FVII polypeptide comprise be incorporated at least one be selected from down the group locational cysteine residues: S12, S23, S43, S45, S52, S53, S60, S67, T83, S103, T106, T108, S111, S119, S126, T128, T130, S147, T185, S214, S222, T255, T267, T307, S320, S333, S336, T370 and S393 (its side chain above 25% is exposed to the surface), described position is selected from especially: S12, S23, S43, S45, S52, S53, S60, S67, S103, S111, S119, S126, S147, S214, S222, S320, S333, S336 and S393 (being occupied by the S residue) more preferably are selected from: S23, S43, S52, S53, S60, S67, T106, T108, S111, S119, S147, S214 and T267 (its side chain above 50% is exposed to the surface) are selected from: S23 especially, S43, S52, S53, S60, S67, S111, S119, S147 and S214 (occupying) by the S residue.
In the other embodiments, cysteine residues is introduced at least one and is selected from above-mentioned arbitrary position and is not the position in tissue factor binding site district.Preferred this position is occupied by T or S residue.For example, the FVII polypeptide comprises introduces at least one locational cysteine residues, and described position is selected from: S12, S23, S45, S52, S53, S67, T83, S103, T106, T108, S111, S119, S126, T128, T130, S147, T185, S214, S222, S232, T233, T238, T239, T255, T267, T293, S320, T324, S333, S336, T370 and S393 (its side chain above 25% is exposed to the surface) are selected from: S12, S23 especially, S45, S52, S53, S67, S103, S111, S119, S126, S147, S214, S222, S232, S233, S320, S333, S336 and S393 (being occupied by the S residue) more preferably are selected from: S23, S52, S53, S67, T106, T108, S111, S119, S147, S214, T238, T267 and T297 (its side chain above 50% is exposed to the surface) are selected from: S23, S52 especially, S53, S67, S111, S119, S147 and S214 (occupying) by the S residue.
In the other embodiments, cysteine residues is introduced at least one and is selected from above-mentioned arbitrary position and is not the position in tissue factor binding site district or avtive spot district.Preferred this position is occupied by T or S residue.For example, the FVII polypeptide comprises introduces at least one locational cysteine residues, and described position is selected from: S12, S23, S45, S52, S53, S67, T83, S103, T106, T108, S111, S119, S126, T128, T130, S147, T185, S214, S222, T255, T267, S320, S333, S336, T370 and S393 (its side chain above 25% is exposed to the surface) are selected from: S12, S23, S45 especially, S52, S53, S67, S103, S111, S119, S126, S147, S214, S222, S320, S333, S336 and S393 (being occupied by the S residue) more preferably are selected from: S23, S52, S53, S67, T106, T108, S111, S119, S147, S214 and T267 (its side chain above 50% is exposed to the surface) are selected from: S23, S52 especially, S53, S67, S111, S119, S147 and S214 (occupying) by the S residue.
Although this respect according to the present invention, the non-polypeptide fractions of conjugate can be any molecule, and when using given coupling method, during as linking group, preferred non-polypeptide fractions is a polymer molecule with cysteine.Polymer molecule can be to be any molecule of describing in the chapters and sections of title with " with the coupling of polymer molecule ", but is preferably selected from linear or ramose Polyethylene Glycol or another polyalkylene oxide.Polymer molecule is PEG in the embodiment, as VS-PEG.Coupling between polypeptide and the polymer can be finished in any suitable manner, as being to describe in the chapters and sections of title with " with the coupling of polymer molecule ", for example uses one-step method or related multistep processes in described chapters and sections.When FVII or FVIIa polypeptide only comprise one can link coupled cysteine residues the time, preferably with have about 5kDa to about 20kDa molecular weight (as from about 10kDa to about 20kDa, be about 5kDa as molecular weight, be about 10kDa, be about 12kDa, be about 15kDa, or be about 20kDa) non-polypeptide fractions coupling, perhaps directly coupling or by the indirect coupling of low-molecular weight polymer (pressing the disclosed method of WO99/55377).When conjugate comprises two or morely can link coupled cysteine residues the time, these non-polypeptide fractions molecular weight separately is generally about 5 to about 10kDa, 5kDa or about 10kDa according to appointment.
Should be appreciated that the described any amino acid whose change in this part, especially replace, can make up with the described amino acid change of any other part, the preferred openly described specific replacement of other parts of specific amino acids change herein comprises the introducing and/or the removal of glycosylation site.
Conjugate of the present invention, wherein non-polypeptide fractions are to have aspartic acid or the glutamic acid molecule as linking group
In another embodiment of the invention, non-polypeptide fractions with aspartic acid or glutamic acid as linking group, be that conjugate of the present invention comprises at least one and the link coupled non-polypeptide of polypeptid covalence, described amino acid sequence of polypeptide is different from wild type FVII shown in the SEQ ID NO:1 or FVIIa sequence part is, introduces or remove at least one asparagicacid residue and/or at least one glutaminic acid residue.
In another embodiment, the aminoacid sequence of FVII or FVIIa polypeptide portion is with different shown in the SEQ IDNO:1, difference is to introduce at least one asparagicacid residue and/or glutaminic acid residue, as 1-15 asparagicacid residue and/or glutaminic acid residue, 1-10 particularly, a 1-6 or 2-4 asparagicacid residue and/or glutaminic acid residue are preferably introduced by replacing.
The position that can introduce asparagicacid residue or glutaminic acid residue includes, but are not limited to above-mentioned proteolysis site, or near it.
Therefore, in one embodiment of the invention, preferably be selected from: I30D/E, K32 D/E by replacing asparagicacid residue and/or the glutaminic acid residue introduced, A34 D/E, T37 D/E, K38 D/E, W41D/E, Y44 D/E, S45 D/E, D46 C, L141 D/E, E142 D/E, K143 D/E, R144 D/E, L288 D/E, R290 D/E, G291 D/E, A292 D/E, Q313 D/E, S314 D/E, R315 D/E, K316 D/E, V317 D/E, L390 D/E, M391 D/E, R392 D/E, S393 D/E, P395 D/E, R396 D/E, P397 D/E, G398 D/E, V399 D/E, L401 D/E, R402 D/E, A403 D/E, P404 D/E or its combination are selected from following group or its combination especially: K32 D/E, Y44 D/E, K143D/E, R290 D/E, R315 D/E, K341 D/E, R392 D/E, R396 D/E, R402 D/E.
Except above-mentioned replacement, can comprise the removal of at least one asparagicacid residue and/or glutaminic acid residue according to the polypeptide of above embodiment conjugate, preferably by replacing.
Because the relative high-load of lysine residue in the parental generation polypeptide thinks that preferred at least one aspartic acid or glutaminic acid residue should be removed, particularly by replacing, to avoid the excessive coupling of non-polypeptide fractions.
Therefore, in the embodiment, the aminoacid sequence of the FVII of conjugate or FVIIa polypeptide portion is with different shown in the SEQ ID NO:1, difference is to remove at least one aspartic acid or glutaminic acid residue, as 1-15 aspartic acid or glutaminic acid residue, 1-10 particularly, a 1-6 or 2-4 aspartic acid or glutaminic acid residue are preferably removed by replacing.For example, preferably be selected from: D33, D46, D48, E77, E82 by replacing aspartic acid or the glutaminic acid residue removed, D86, D87, E94, E99, D104, E116, D123, E132, E142, E163, D196, E210, D212, E215, D217, D219, E220, D256, E265, E270, D289, E296, D309, D319, E325, D334, D338, D343, E385, E394 or its combination.
Should be understood that at least one aspartic acid or the glutaminic acid residue introduced to parental generation molecule predetermined site, it is especially preferred making up with the removal of at least one above-mentioned aspartic acid or glutaminic acid residue.Therefore, in embodiment preferred of the present invention, the aminoacid sequence of conjugate FVII or FVIIa polypeptide portion is with different shown in the SEQ ID NO:1, and difference is that at least one aspartic acid or glutaminic acid residue are removed (preferably by substituting) and at least one aspartic acid or glutaminic acid residue and are introduced into (preferably by substituting.
This one side according to the present invention, have aspartic acid group or glutamic acid group as the non-polypeptide fractions of the conjugate of its linking group, it can be any non-polypeptide fractions with this specific character, preferred non-polypeptide fractions is polymer molecule or organic derivatizing agent, particularly polymer molecule, and the preparation of conjugate such as Sakane and Pardridge Pharmaceutical Research, Vol.14, No.8,1997, pp1085-1091.
Should be appreciated that the described any amino acid change in described this part, especially replace, can make up with the described amino acid change of any other part, the openly specific replacement of other parts of amino acid change comprises the introducing and/or the removal of glycosylation site especially herein.
Conjugate of the present invention, wherein non-polypeptide fractions is a saccharic composition
In another embodiment of the present invention, inserted and/or removed the linking group of saccharic composition, as glycosylation site, particularly intravital glycosylation site.
Preferably, conjugate of the present invention comprises at least one saccharic composition that is connected with polypeptid covalence, the difference of wild type FVII or FVIIa sequence shown in wherein said amino acid sequence of polypeptide and the SEQ ID NO:1 is, wherein the glycosylation site that produces of at least one non-natural is introduced into and/or the glycosylation site of at least one natural generation is removed.Especially, the introducing of the removal of the glycosylation site of natural generation and non-natural glycosylation site, or the removal of non-natural glycosylation site combination.The glycosylation site of introducing can be O-glycosylation site or N-glycosylation site.Preferred glycosylation site is O-glycosylation site or the interior N-glycosylation site of body in the body, N-glycosylation site in the special preferred body.
The term " glycosylation site of natural generation " that is used for this paper is included in N145, N322, the glycosylation site of S52 and S60 position.Same, term " O-glycosylation site in the body of natural generation " comprises the glycosylation site of S52 and S60 position, and term " N-glycosylation site in the body of natural generation " comprises the glycosylation site of N145 and N322 position.
Usually, sequence difference shown in FVII or FVIIa amino acid sequence of polypeptide and the SEQ ID NO:1 is, introduce the glycosylation site (as N-glycosylation site in the body of at least one non-natural generation) that at least one non-natural produces, glycosylation site (as N-glycosylation site in the body of 1-15 non-natural generation) as 1-15 non-natural generation, 1-10 particularly, 1-6, or the glycosylation site of 2-4 non-natural generation is (as 1-10,1-6 or 2-4 the interior N-glycosylation site of body that non-natural produces), preferably introduce by replacing.
Be interpreted as preparation conjugate (wherein said conjugate polypeptide comprises one or more glycosylation sites), described polypeptide must be expressed in that sugar (oligosaccharide) component is connected in the host cell of glycosylation site, perhaps in the external glycosylation of carrying out.Can in following " with the coupling of saccharic composition " joint, example be arranged further by glycosylated host cell.
In the embodiment of this respect, glycosylation site is introduced into the position that is occupied by the amino acid residue that is exposed to this molecular surface in parental generation FVII or the FVIIa molecule in the body, preferably surpass 25% side chain and be exposed to those amino acid residues in the solvent, particularly surpass 50% side chain and be exposed to those (these positions are determined in the method chapters and sections in the text) in the solvent.Introduce the N-glycosylation site then, used mode makes the N-residue in described site be arranged in described position.Similarly, introduce the O-glycosylation site and be positioned at described position so that form the S or the T residue in this site.Described position comprises K32S/T, I42S/T, Y44S/T, K143S/T, R290S/T, R315S/T, K341S/T, R392S/T, R396S/T, R402S/T or its combination.
During the N-glycosylation, glycosylation site in the body can be introduced only needs a sudden change just can create the position (that is, create required any other amino acid residue in functional glycosyl site and be present in this intramolecularly) in described site.
In other words, the preferably such conjugate of conjugate of the present invention, wherein the difference of polypeptid acid sequence and SEQ ID NO:1 is, the N-X ' of at least one natural generation-X sequence is replaced by N-X '-S or N-X '-T sequence, wherein X ' is any aminoacid except that proline (P), and X is any aminoacid except that serine (S) and threonine (T).
Similarly, the preferably such conjugate of conjugate of the present invention, wherein the difference of polypeptid acid sequence and SEQ ID NO:1 is, X-X '-the S of natural generation or X-X '-T sequence are replaced by N-X '-S or N-X '-T sequence among at least one SEQ ID NO:1, wherein X ' is any aminoacid except that proline (P), and X is any aminoacid except that N.
Create this class of N-glycosylation site replaces in the body particular instance and comprise the replacement that is selected from following group or its combination: F4S/T, P10N, Q21N, W41N, S43N, A51N, G58N, L65N, G59S/T, E82S/T, N95S/T, G97S/T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N, T128N, A175S/T, G179N, I186S/T, V188N, R202S/T, I205S/T, D212N, E220N, i230N, P231N, P236N, G237N, V253N, E265N, T267N, E270N, R277N, L280N, G291N, P303S/T, L305N, Q312N, G318N, G331N, D334N, K337N, G342N, H348N, R353N, Y357N, 1361N, V376N, R379N, M391N.Preferred described replacement is selected from following group: F4S/T, P10N, Q21N, W41N, A51N, G58N, G59S/T, N95S/T, G97S/T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N, T128N, A175S/T, I186S/T, V188N, R202S/T, I205S/T, D212N, E220N, V253N, E265N, T267N, E270N, L280N, G291N, P303S/T, G318N, G331N, D334N, K337N, R353N, Y357N, M391N or its combination.
Alternate, glycosylation site is introduced by lysine residue or arginine residues position occupied in the body, is preferably occupied by lysine, and particularly the S of the N-residue of N-glycosylation site or O-glycosylation site or T residue replace described lysine residue.
In other words, the preferably such conjugate of conjugate of the present invention, wherein the difference of amino acid sequence of polypeptide and SEQ ID NO:1 is, K-X '-X the sequence of natural generation or R-X '-X sequence among at least one SEQ ID NO:1, preferred K-X '-X sequence, replaced by N-X '-S or N-X '-T sequence, wherein X ' is any aminoacid except that proline (P), and X is any aminoacid except that serine (S) and threonine (T).
Creating in the body example of the replacement of N-glycosylation site comprises and is selected from following group replacement: K32N+A34S/T, K38N+F40S/T, K62N+Q64S/T, K85N+D87S/T, K137N+P139S/T, K143N+N145S/T, K148N+Q149S/T, K161N+E163S/T, K197N+K199S/T, K199N+W201S/T, K316N+G318S/T, K337N, K341N+D343S/T, K389N+M391S/T or its combination.
In the embodiment of above-mentioned glycosylation aspect, glycosylation site, especially N-glycosylation site can be introduced into above-mentioned proteolysis site, or near it (referring to " conjugate of the present invention " chapters and sections).
Therefore, creating in the body particular instance of the replacement of N-glycosylation site comprises and is selected from following group replacement: K32N+A34S/T, F31N+D33S/T, I30N+K32S/T, A34N+R36S/T, K38N+F40S/T, T37N+L39S/T, R36N+K38S/T, L39N+W41S/T, F40N+I42S/T, W41N, I42N+Y44S/T, S43N, Y44N+D46S/T, S45N+G47S/T, D46N+D4SS/T, G47N+Q49S/T, K143N+N145S/T, E142N+R144S/T, L141N+K143S/T, I140N+E142S/T, R144N+A146S/T, A146N+K148S/T, S147N+P149S/T, R290N+A292S/T, D289N+G291S/T, L288N+R290S/T, L287N+D289S/T, G291N, A292N+A294S/T, T293N+L295S/T, R315N+V317S/T, S314N+K316S/T, Q313N+R315S/T, Q312N, K316N+G318S/T, V317N+D319S/T, G318N, K341N+D343S/T, S339N+K341S/T, G342N, D343N+G345S/T, R392N+E394S/T, M391N, L390N+R392S/T, K389N+M391S/T, S393N+P395S/T, E394N+R396S/T, P395N+P397S/T, R396N+G398S/T, P397N+V399S/T, G398N+L400S/T, V399N+L401S/T, L400N+R402S/T, L401N+A403S/T, R402N+P404S/T, A403N+F405S/T, P404N+P406S/T
Or its combination, as K143N+N145S/T+R315N+V317S/T.Preferably, described replacement is selected from following group:
K32N+A34S/T，K38N+F40S/T，Y44N+D46S/T，K143N+N145S/T，R290N+A292S/T，R315N+V317S/T，K341N+D343S/T，R392N+E394S/T，R396N+G398S/T，R402N+P404S/T
Or its combination, as K143N+N145S/T+R315N+V317S/T.More preferably, described replacement is selected from: K32N+A34T, K38N+F40T, Y44N+D46T, K143N+N145T, R290N+A292T, R315N+V317T, 341N+D343T, R392N+E394T, R396N+G398T, R402N+P404T
Or its combination, particularly K143N+N145T+R315N+V317T.
In another embodiment of the invention, glycosylation site is introduced into such position in the preferred body, and it is formative tissue factor bound fraction neither, does not also form avtive spot district part and the ridge of the avtive spot engagement groove that defines herein.Can imagine that this glycosylation variant mainly belongs to the active conjugate of this paper definition before.
Therefore, creating in the body particular instance of the replacement of N-glycosylation site comprises and is selected from following group replacement:
K32N+A34S/T，I30N+K32S/T，A34N+R36S/T，K38N+F40S/T，T37N+L39S/T，W41N，Y44N+D46S/T，S45N+G47S/T，D46N+D48S/T，G47N+Q49S/T，K143N+N145S/T，E142N+R144S/T，L141N+K143S/T，I140N+E142S/T，R144N+A146S/T，A146N+K148S/T，S147N+P149S/T，L288N+R290S/T，L287N+D289S/T，R315N+V317S/T，S314N+K316S/T，K316N+G318S/T，V317N+D319S/T，G318N，R392N+E394S/T，M391N，L390N+R392S/T，K389N+M391S/T，S393N+P395S/T，E394N+R396S/T，P395N+P397S/T，R396N+G398S/T，P397N+V399S/T，G398N+L400S/T，V399N+L401S/T，L401N+A403S/T，R402N+P404S/T，A403N+F405S/T，P404N+P406S/T
Or its combination, as K143N+N145S/T+R315N+V317S/T.Preferably, described replacement is selected from following group:
K32N+A34S/T，K38N+F40S/T，Y44N+D46S/T，K143N+N145S/T，R315N+V317S/T，R392N+E394S/T，R396N+G398S/T，R402N+P404S/T
And combination, as K143N+N145S/T+R315N+V317S/T.More preferably, described replacement is selected from:
K32N+A34T，K38N+F40T，Y44N+D46T，K143N+N145T，R315N+y317T，R392N+E394T，R396N+G398T，R402N+P404T
Or its combination, particularly K143N+N145T+R315N+V317T.
In another embodiment of the invention, glycosylation site is introduced into such position in the preferred body, and it is formative tissue factor bound fraction not, but it forms the part in avtive spot district and the ridge of the avtive spot engagement groove that defines herein.Can imagine that this glycosylation variant mainly belongs to the non-activity conjugate of this paper definition before.
So, create in this body particular instance of the replacement of N-glycosylation site and comprise and be selected from following replacement: I153N+G155S/T, Q167N+L169S/T, V168N+L170S/T, L169N+L171S/T, L170N+V172S/T, L171N+N173S/T, A175S/T, A175N+L177S/T, L177N+G179S/T, G179N, G180N+L182S/T, T181N+I183S/T, V188N, V189N+A191S/T, S190N+A192S/T, A191N+H193S/T, H193N+F195S/T, F195N+K197S/T, D196N+I198S/T, K197N+K199S/T, I198N+N200S/T, K199N+W201S/T, W201N+N203S/T, R202S/T, I205S/T, V228N+I230S/T, I229N+P231S/T, I230N, P231N, S232N+Y234S/T, T233N+V235S/T, Y234N+P236S/T, V235N+G237S/T, P236N, G237N, T238N+N240S/T, T239N+H241S/T, H241N+I243S/T, D242S/T, I243N+L245S/T, A244N+L246S/T, L245N+R247S/T, L246N+L246S/T, V281N+G283S/T, S282N+W284S/T, G283N+G285S/T, W284N+Q286S/T, G285N+L287S/T, Q286N+L288S/T, D289N+G291S/T, R290N+A292S/T, G291N, A292N+A294S/T, T293N+L295S/T, P321N+I323S/T, T324N+Y326S/T, E325N+M327S/T, Y326N+F327S/T, F328N+A330S/T, S339N+K341S/T, K341N+D343S/T, G342N+S344S/T, D343N+G345S/T, S344N+G346S/T, G345N+P347S/T, P347N+A349S/T, H348N, L358N+G360S/T, T359N+I361S/T, G360N+V362S/T, I361N, V362N+W364S/T, S363N+G365S/T, W364N+Q366S/T, G365N+G367S/T, T370N+G372S/T, V376N, Y377N+R379S/T, T378N+V380S/T, R379N, V380N+Q382S/T, Q382N+I384S/T, Y383N+E385S/T, W386N+Q388S/T, L387N+K389S/T, L400N+R402S/T
And combination.Be preferably selected from following replacement: D289N+G291S/T, R290N+A292S/T, G291N, A292N+A294S/T, T293N+L295S/T, S339N+K341S/T, K341N+D343S/T, G342N+S344S/T, D343N+G345S/T,
And combination, more preferably be selected from following replacement: D289N+G291T, R290N+A292T, G291N, A292N+A294T, T293N+L295T, S339N+K341T, K341N+D343T, G342N+S344T, D343N+G345T,
And combination.
Except saccharic composition, the conjugate that this respect illustrates in these chapters and sections according to the present invention can contain other non-polypeptide fractions, particularly polymer molecule, and as described herein, it is coupled on one or more linking groups of conjugate polypeptide portion.
Should understand any amino acid change specific in these chapters and sections, especially replace, can change (especially replacing) combination with the specific amino acids that illustrates in other parts of this paper.
For example, (described variant has at least one glycosylation site that is introduced into and/or removes to disclosed any glycosylation variant in these chapters and sections, as comprise the replacement of R315N+V317T and/or K143N+N145T), can be further and a polymer molecule, as PEG, or any other non-polypeptide fractions coupling.For this reason, can be by using existing linking group among FVII or the FVIIa, or be introduced into and/or remove linking group, make that especially can be used for link coupled linking group adds up to 1-6,3-4 or 1,2,3,4,5 particularly, or 6 and obtain conjugate.
Preferably, in conjugate of the present invention (wherein FVII or FVIIa polypeptide comprise two glycosylation sites), the quantity of non-polypeptide fractions and molecular weight be through selection so that add total molecular weight behind the non-polypeptide fractions in the scope of 5-25kDa, as in the 10-25kDa scope, particularly about 5kDa, 12kDa, 15kDa or 20kDa.
The non-activity conjugate
Conjugate of the present invention can cause inactivation by removing at least one amino acid residue, and the position that described aminoacid occupies is selected from the R152 of SEQ ID NO:1, I153, S344, D242 and H193.This removal can realize by replacing or lacking one or more above-mentioned amino acid residues.Preferably, this removal is to realize by replacing, and especially realizes by conservative the replacement.Accordingly, used herein non-activity FVII or FVIIa polypeptide can comprise one or more following replacements: R152X, I153X, and S344X, D242X or H193X, wherein X is any amino acid residue, the amino acid residue that preferably causes conservative to replace.For example, the FVII of non-activity or FVIIa polypeptide comprise the R152X sudden change, and wherein X is any amino acid residue (because lysine forms the part in Partial Protein enzymolysis site) except lysine.Other examples that specificity replaces comprise I153A/V/L; S344T/A/G/Y; D242E/A and/or H193R/A.
Alternate, active FVII or FVIIa may be because of the carbamylations of I153 alpha amino acid group, or by with described polypeptide and compound its inactivation that makes of serpin.Suitable serine Profilin, for example be selected from organic phosphorus compound, sulfanylfluoride, halomethyl ketone peptide, preferred red sulfonic acid-D-phenylalanyl-prolyl-arginine chloromethyl ketone, red sulfonic acid-glutamic acid-glutamic acid-arginine chloromethyl ketone, red sulfonic acid-phenylalanine-phenylalanine-arginine chloromethyl ketone, or phenylalanine-phenylalanine-arginine chloromethyl ketone, or azapeptide.
Conjugate also can cause inactivation by introducing at least one glycosylation site in selected position, so that follow-up glycosylation makes the conjugate inactivation.
As above described in the decline of " conjugate of the present invention; wherein non-polypeptide fractions is a saccharic composition " chapters and sections, preferred glycosylation site is introduced into such site, its not the formative tissue factor a part but form the part in avtive spot district and the ridge of the avtive spot engagement groove of this paper definition.The preferred specific examples that replaces provides in " conjugate of the present invention, wherein non-polypeptide fractions is a saccharic composition " chapters and sections.
The non-polypeptide fractions of conjugate of the present invention
As mentioned above, the non-polypeptide fractions of conjugate of the present invention is preferably selected from polymer molecule, lipophilic compound, saccharic composition (by glycosylated mode in the body) and organic derivation agent.The polypeptide portion that all these materials all can be conjugate provides required character, particularly increases in the function gonosome half life and/or increases the blood plasma half life.The polypeptide portion of conjugate can be only and one type non-polypeptide fractions coupling, but also can with two or more dissimilar non-polypeptide fractions couplings, for example, with polymer molecule and saccharic composition coupling, with lipophilic group and saccharic composition coupling, with organic derivation agent and saccharic composition coupling, with lipophilic group and polymer molecule coupling etc.Can simultaneously or carry out in order with two or more dissimilar non-polypeptide fractions couplings.
The method for preparing conjugate of the present invention
In following chapters and sections " with the coupling of lipophilic compound ", the coupling with the non-polypeptide of each specified type is described in " with the coupling of polymer molecule ", " with the coupling of saccharic composition " and " with the coupling of organic derivation agent ".Generally, polypeptide conjugate of the present invention is by helping cultivating proper host cell under the described expression of polypeptides condition, obtain described polypeptide, wherein a) described polypeptide comprises at least one N-or O-glycosylation site, described host cell is to carry out glycosylated eukaryotic cell in the body, and/or b) described polypeptide can the non-polypeptide fractions of external coupling.
Should be understood that to make the quantity of gained molecule the size of this quasi-molecule and form (as linearity or branch), and the connection site each side the best on described polypeptide at the non-polypeptide fractions that is connected to link coupled design.The molecular weight of the non-polypeptide fractions that uses can, select as the effect that reaches based on hope.For example, if link coupled main purpose is to obtain to have high-molecular weight conjugate (as reducing renal clearance), want the coupling non-polypeptide fractions of few high molecular of trying one's best usually, with the molecular weight that obtains to expect.If wish to obtain covering of height, can use capacity the non-polypeptide fractions of low-molecular-weight (as molecular weight from about 300Da to about 5kDa, as molecular weight from 300Da to 2kDa), effectively covering all or most proteolysis site, or other easy ruined sites in the described polypeptide.
Coupling with lipophilic compound
Polypeptide and lipophilic compound can directly or pass through to use joint coupling each other.Lipophilic compound can be natural chemical compound such as saturated or unsaturated fatty acid, fatty acid diketone, terpenes, prostaglandin, vitamin, carotenoid or steroid hormone, perhaps synthetic chemical compound such as carbonic acid, alcohol, amine and have one or more alkyl-, aryl-, alkenyl-sulfonic acid or other polynary unsaturated compound.Can be by methods known in the art, as press Bodanszky at Peptide Synthesis, John Wiley, New York, 1976 and the described method of WO96/12505 carry out coupling (optional connect) between polypeptide and the lipophilic compound by joint.
With the polymer molecule coupling, comprise the coupling of polymer molecule and polypeptide N-terminal
With the link coupled polymer molecule of polypeptide can be any suitable polymers molecule, as natural or synthetic homopolymer or heteropolymer, the molecular weight ranges of common described molecule is about 300-100000Da, 500-20000Da according to appointment, be more preferably 500-15000Da, even preferred scope is about 2-15kDa, is about 3-10kDa as scope.When term " about " used herein related to a certain molecular weight, " pact " just represented about mean molecule quantity, and reflected certain molecular weight distribution is promptly arranged such fact usually in certain polymer product.
The example of homopolymer comprise polyalcohols (that is, poly--OH), polyamine (that is, poly--NH2) and polycarboxylic acids (that is, gather-COOH).Heteropolymer is to comprise different coupling group, as the polymer of oh group and amino group.
The example of suitable polymers molecule comprises and is selected from following polymer molecule: polyalkylene oxide (PAO), comprise poly alkylene glycol (PAG), as Polyethylene Glycol (PEG) and polypropylene glycol (PPG), ramose PEG, polyvinyl alcohol (PVA), Merlon, polyvinylpyrrolidone, polyethylene is maleic anhydride altogether, polystyrene is maleic anhydride altogether, and glucosan comprises that Sensor Chip CM 5 or any other are suitable for reducing immunogenicity and/or increase half life in the function gonosome and/or the biopolymer of serum half life.Another example of polymer molecule is human albumin or another abundant plasma protein.In general, polyalkylene glycol-derived polymers is biocompatible, nontoxic, no antigen, non-immunogenicity, has various water solublity characteristics and be easy to secrete from the biology of living.
PEG is preferred polymer molecule because its with, for example polysaccharide such as glucosan compare only have minute quantity can be crosslinked reactive group.Interested especially is single function PEG, as mono methoxy polyethylene glycol (mPEG) because its link coupled chemistry simple relatively (only reactive group can be used for polysaccharide on the linking group coupling).Therefore, crosslinked danger is eliminated, and the gained polypeptide conjugate more reaction of homogeneous and polymer molecule and polypeptide is easier to control.
For polymer molecule is combined with polypeptid covalence, the hydroxyl terminal groups of polymer molecule is necessary for activated form, promptly has reactive functional group (example comprises primary amine group, hydrazides (HZ), sulfydryl, succinate (SUC), succinimido succinate (SS), succinimido succinamide (SSA), succinyl phosphorons amino propyl acid ester (SPA), succinimido carboxy methylation thing (SCM), benzotriazole carbonic ester (BTC), N-hydroxy-succinamide (NHS), aldehyde, nitrophenyl carbonate (NPC) and tresylate (TRES)).Suitable activated polymer molecule has commodity for example can derive from ShearwaterPolymer, Inc., Huntsville, AL, USA or derive from PolyMASC Pharmaceuticals plc, UK.
Perhaps, polymer molecule can for example, activate described in WO 90/13540 by conventional method known in the art.The particular instance of employed activated linearity or ramose polymer molecule is described in Shearwater Polymers among the present invention, Inc.1997 and 2000 Catalogs (FunctionalizedBiocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycoland Derivatives is incorporated herein for reference).
The particular instance of activated PEG polymer comprises following linear PEGs:NHS-PEG (SPA-PEG for example, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG and side chain PEG such as PEG2-NHS and US 5,932,462 and US 5, in 643,575 disclosed those, with two with reference to be incorporated herein for reference.In addition, be incorporated herein and disclose useful polymer molecule and/or PEGization chemical process: US 5,824,778 in the following publication for reference, US 5,476,653, and WO 97/32607, EP 229,108, and EP 402,378, US 4,902,502, and US 5,281,698, US5,122,614, US 5,219, and 564, WO 92/16555, and WO 94/04193, and WO 94/14758, and WO 94/17039, WO94/18247, WO 94/28024, and WO 95/00162, and WO 95/11924, WO 95/13090, and WO 95/33490, WO96/00080, and WO 97/18832, WO 98/41562, and WO 98/48837, and WO 99/32134, and WO 99/32139, WO99/32140, WO 96/40791, and WO 98/32466, and WO 95/06058, EP 439 508, and WO 97/03106, WO96/21469, and WO 95/13312, EP 921 131, and US 5,736,625, WO 98/05363, and EP 809 996, US5,629,384, WO 96/41813, and WO 96/07670, and US 5,473,034, US 5,516,673, EP 605 963, US5,382,657, EP 510 356, and EP 400 472, EP 183 503 and EP 154 316.
The coupling of polypeptide and activated polymer molecule can be by using any conventional method, for example, undertaken by following list of references described (also having described the proper method that activates polymer molecule in the described list of references): R.F.Taylor, (1991), ＂ Protein immobilisation.Fundamental and applications ＂, Marcel Dekker, N.Y.; S.S.Wong, (1992), ＂ Chemistry of Protein Conjugationand Crosslinking ＂, CRC Press, Boca Raton; G.T.Hermanson et al., (1993), ＂ Immobilized Affinity Ligand Techniques ＂, Academic Press, N.Y.).The functional group that the technical staff can know linking group (the above example that provided) according to described polypeptide and polymer (for example, be amino, hydroxyl, carboxyl, aldehyde, sulfydryl, butanimide, maleimide, vinysulfone or halogenated acetic acid salt) use Activiation method and/or coupling chemistry.PEGization can directly carry out with all the available groups (that is, being exposed to the linking group on polypeptide surface) on the polypeptide coupling or can with one or more specific linking groups as, the direct coupling of N-terminal amino group (describing) as US5985265.And, coupling can in a step, finish or with the multistep mode (as, described in the WO99/55377) finish.
Should understand PEG turn into need be designed to make the gained molecule bonded PEG molecule number, these bulks of molecule and form (as, be linear or ramose), and in peptide molecule in conjunction with position each side the bests of these molecules.For example, the effect that can reach is as required selected the molecular weight of employed polymer.For example, if link coupled main purpose be produce and to have high-molecular weight conjugate (as, reduce the removing of kidney), it is desirable to the least possible heavy polymer molecule of coupling usually to obtain the desired molecule amount.When needs are highly covered, low-molecular weight polymer that can be by using sufficient amount (as, molecular weight is about 300Da-5kDa) obtain, can cover the destructive site that is subject to of all or most proteolysis site or described polypeptide in the polypeptide so effectively.For example, can use 2-8, the polymer as 3-6.
Only with protein on during a linking group coupling (as N-terminal amino group group), advantageously linear or ramose polymer has high molecular, preferably about 10-25kDa, 15-25kDa according to appointment, for example about 20kDa.
In general, the coupling condition of polymer is to make all available polymer linking group and reacted polymer molecules.This can realize for the suitable molar excess of described polypeptide by making described polymer phase.Usually, the mol ratio of activated polymer molecule and polypeptide is 1000-1,200-1, or about 100-1 according to appointment.Also possibility is lower slightly in order to obtain the described in some cases mol ratio of optimum response, according to appointment 50-1,10-1 or 5-1.
The present invention also plans by joint polymer molecule and polypeptide coupling.Suitable joint is that the technical staff is known.Preferred examples is cyanuric chloride (Abuchowski etc., (1977), J.Biol.Chem., 252,3578-3581; US4179337; People such as Shafer (1986), J.Polym.Sci.Polym.Chem.Ed., 24,375-378).
After the coupling, as sealing residual activated polymer molecule in the reactant mixture, and remove the polymer molecule of the inactivation that is produced by suitable method by primary amine is joined by methods known in the art.
Should understand based on various conditions (as amino acid sequence of polypeptide, the character of the active PEG chemical compound that uses and specific PEGization condition, the mol ratio that comprises PEG and polypeptide), may obtain PEGization in various degree, the common higher mole ratio of the PEGization of higher degree from PEG and polypeptide.Yet, derive from the PEGization polypeptide of any given PEGization process, comprise the random distribution of the slightly different polypeptide conjugate of PEGization degree usually.
One embodiment of the invention, polypeptide conjugate of the present invention comprise one with the covalently bound polymer molecule of the terminal A1 of wild type FVII shown in the SEQ ID NO:1 or FVIIaN, wherein said polymer molecule is the unique polymer molecule that is connected with described polypeptide.Preferred this peptide species conjugate comprises the single PEG molecule that is connected with this polypeptide N-terminal, and does not have other PEG molecules.Special preferred molecular weight is at least about 5kDa, especially about 10-25kDa, 15-25kDa, for example linearity or the ramose PEG molecule of about 20kDa according to appointment.Polypeptide conjugate according to this embodiment can further comprise one or more saccharic compositions, and described saccharic composition is connected in glycosylation site that described polypeptide N-connects or that O-connects, or connects by external glycosylation.
In another embodiment of the invention, polypeptide conjugate of the present invention comprises polymer molecule, wild type FVII or FVIIa N-terminal A1 and N-terminal I153 are covalently bound shown in described polymer molecule and the SEQ ID NO:1, and described polymer molecule is the unique polymer molecule that is connected with described polypeptide.Preferred this peptide species conjugate comprises the single PEG molecule that is connected with this polypeptide N-terminal, and does not have other PEG molecules.Special preferred molecular weight is at least about 5kDa, especially about 10-25kDa, and as 15-25kDa, for example about 20kDa is preferred.Polypeptide conjugate according to this embodiment can further comprise one or more saccharic compositions, and described saccharic composition is connected in glycosylation site that described polypeptide N-connects or that O-connects, or connects by external glycosylation.
Polymer (as the PEG molecule) optionally is coupled in the method for polypeptide N-end, and disclosed method is preferred among the US5985265.Described method comprise standard reductive alkylation (polypeptide N-terminal amino group group with contain the polypeptide of aldehyde radical, as aldehyde radicalization-PEG, there being Reducing agent, as NaCNBH3, condition under react).Described method is utilized the differential responses that can be used for deutero-different primary amino radical groups (lysine and N-end compare) in the polypeptide, obtains the derivant of the plurality of optional of described polypeptide, and its N-terminal carboxyl groups comprises polymer molecule, as hydroformylation PEG.Described being reflected under the pH value carried out, and described pH value allowance utilizes the pKa difference of the epsilon-amino group and the polypeptide N-terminal residue α amino group of lysine residue.In order to obtain this differential responses, described reaction is carried out under mild acid conditions usually.Suitable pH value scope comprises pH4.5-7, as pH4.5-6, and as pH5-6, particularly about pH5.
In other particular, polypeptide conjugate of the present invention comprises a PEG molecule, described PEG molecule is connected on each lysine residue that can carry out PEGization in the polypeptide, especially linear or ramose PEG molecule, as the about 1-15kDa of molecular weight, typical about 2-12kDa, 3-10kDa, 5kDa or 6kDa according to appointment according to appointment.
In other embodiments, polypeptide conjugate of the present invention comprises the PEG molecule, and described PEG molecule is connected on each lysine residue that can carry out PEGization in the polypeptide, also is connected on the amino acid residue of polypeptide N-end simultaneously.
Carbohydrate ingredient (as glucosan) with as described in the polypeptide amino acid residue also can use people's such as WO87/05330 and Aplin CRC Crit Rev.Biochem. at external covalent coupling, pp.259-306, the method for describing in 1981.The external coupling of carbohydrate ingredient or PEG and protein bound type and peptide conjunction type Gln-residue also can be undertaken by transglutaminase (TGases).Transglutaminase with a kind of mode catalysis donor amino group of so-called cross-linking reaction transfer to albumen-and peptide-bonded Gln residue on.The donor amino group can be albumen-or peptide-bonded, as the ε amino group in the lysine residue, or also can be the part of little or big organic molecule.In transglutaminase-catalyzed crosslinked as the little organic molecule such as the putrescine (1,4 diaminobutane) of amino donor.In transglutaminase-catalyzed crosslinked as the big organic molecule of amino donor as contain amino-PEG (Sato etc., 1996, Biochemistry 35,13072-13080).
Usually transglutaminase is the enzyme of high special, be not all Gln residues that are exposed to protein surface all can be used for transglutaminase-catalyzed contain the crosslinked of amino material.Having only minority Gln residue on the contrary is the natural substrate of transglutaminase, but still unknowable as the definite parameter of the transglutaminase substrate that is fit to for control Gln residue.Therefore, in order to make the cross-linking reaction sensitivity of albumen to transglutaminase-catalyzed, frequent prerequisite is at one section aminoacid sequence of position increase easily, the known substrate that can be used as transglutaminase well of described aminoacid sequence.Known several aminoacid sequence can be the substrate of transglutaminase, or comprises the substrate of transglutaminase, as the P material, and elafin, Fibrinogen, fibronectin, α 2-plasmin inhibitor, alpha-casein, and beta-casein.
Coupling with saccharic composition
For glycosylation in the body of finishing FVII molecule (comprising one or more glycosylation sites), the nucleotide sequence of coded polypeptide must be inserted among the glycosylated eukaryotic expression host.Expression host cell can be selected from fungus (filamentous fungi or yeast), insecticide or zooblast, or is selected from transgenic plant cells.In one embodiment, host cell is a mammalian cell, as Chinese hamster ovary celI, bhk cell or HEK cell, as the HEK293 cell, or insect cell, as the SF9 cell, perhaps yeast cells such as saccharomyces cerevisiae (Saccharomyces Cerevisiae) or finish red saccharomyces pastorianus (Pichia pastoris) or any host cell that hereinafter further describes.
Coupling with organic derivating agent
The covalent modification of described polypeptide can be by polypeptide one or more linking groups and organic derivating agent react and carry out.Suitable derivating agent and method are known in the art.For example, the cysteinyl-residue is usually with (halogenated acetic acids ester (with corresponding amine) produces carboxymethyl or carboxy and amide groups methyl-derivatives as monoxone or chloroacetamide reaction.The cysteinyl-residue also can be with the bromine trifluoroacetone, (bromo-((4-imidazole radicals) propanoic acid, chloracetyl phosphate ester, N-alkyl maleimide, 3-nitro-2-pyridine disulphide, methyl 2-pyridine disulphide, pCMBA salt, 2-chloromercuri-4-nitrophenols or chloro-7-nitro benzo-2--1, derive by 3-diazole reaction.The histidyl-residue is derived by reacting under pH5.5-7.0 with the diethyl pyrocarbonate, because this reagent is specific to the histidyl-side chain.The PBPB thing can be used for deriving; This reaction is preferably reacted under pH6.0 in 0.1M cacodylic acid sodium.Lysyl-and n terminal residue can react with succinic anhydrides or other carboxylic acid anhydrides.Derive with these reagent and to have the effect of the charge reversal that makes the lysyl-residue.Deriving and contain, (other suitable reagent of amino residue comprises imino esters such as methyl picolinimidate, phosphoric acid Vitamin B6, Vitamin B6, chloro borohydrides, trinitro-benzene-sulfonic acid, adjacent methyl-isourea, 2, the catalytic reaction with glyoxylate of 4-pentanedione and transaminase.The arginyl-residue is by modifying with the reaction of one or more conventional reagent, and described reagent comprises phenyl Biformyl, 2,3-diacetyl, 1,2-cyclohexanedione and 1,2,3-indantrione monohydrate.The requirement of deriving of arginine residues is reflected under the alkali condition to be carried out, because the guanidine functional group has high pKa.
In addition, these reagent can react with lysine and arginine guanidine radicals.Carboxyl side group (aspartyl or glutamyl) is by optionally modifying with carbodiimide (R-N=C=N-R ') reaction; wherein R is different alkyl with R '; as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-nitrogen father-in-law-4,4-dimethyl amyl group) carbodiimide.And aspartyl and glutamyl are by being converted into Radix Asparagi acyl amide groups and glutamy amide groups with the ammonium ion reaction.
The sealing in functional site
The excessive coupling of having reported polymer can cause and the link coupled polypeptide active forfeiture of polymer.This problem can by as remove the linking group be positioned at functional site, or reversibility closing function site is capped functional site and eliminates when coupling before coupling.A kind of strategy in back constitutes the further technical scheme of the present invention (preceding a kind of strategy illustrates hereinbefore, as the lysine residue by the contiguous functional site of removal).More particularly, according to second kind of strategy, the coupling between polypeptide and the non-polypeptide fractions be the polypeptide functional site with can in conjunction with the accessory molecule in polypeptide functional site (as can in conjunction with as described in the tissue factor or the serpin of polypeptide functional site) carry out under the condition of sealing.
Preferably, accessory molecule energy specific recognition polypeptide functional site, as receptor, particularly tissue factor, or total length, or the tissue factor of suitable clipped form, or two molecules, one of them is a tissue factor, and another is the inhibitor of a kind of peptide or peptide, and it can be attached to, and (preferred definition is near the catalysis triplet on the catalysis triplet
Figure C01804864D00391
The zone amino acid residue), thereby protect this zone.
In addition, accessory molecule can be an antibody, particularly discerns the monoclonal antibody of FVII polypeptide.Particularly, accessory molecule can be a neutralizing monoclonal antibody.
Polypeptide was interacted with accessory molecule before coupling.Therefore and can not be by non-polypeptide fractions such as polymer-derived this guarantees that the functional site of polypeptide is covered or protected and.Behind the accessory molecule eluting, the conjugate between non-polypeptide fractions and the polypeptide can recover with the functional site that keeps to small part.
Have the polypeptide in the functional site that is closed and the coupling subsequently of polymer, lipophilic compound, saccharic composition, organic derivating agent or any other chemical compound and carry out in a usual manner, as being undertaken by the method described in the chapters and sections of above " with ... coupling ".
Do not consider to be used for to cover the character of the accessory molecule in conjugate polypeptide functional site, it is desirable to that accessory molecule does not conform to or only contain in a spot of and the molecule specify the bonded group of non-polypeptide fractions, wherein will stop the desorbing from the accessory molecule of link coupled polypeptide with the coupling of these groups.Therefore, can be used for the repetition coupling repeatedly with the non-coupling of linking group selectivity and the accessory molecule that exist in the part covered of polypeptide.For example, if non-polypeptide fractions to be polymer molecule such as PEG this have lysine (amino or-terminal amino acid residue be as the branch period of the day from 11 p.m. to 1 a.m of linking group, it is desirable to accessory molecule be substantially free of can be link coupled (amino does not preferably contain any (amino.Therefore, in preferred embodiments, accessory molecule be can with bonded protein in polypeptide functional site or peptide, this protein or peptide do not contain any with specify the non-polypeptide fractions can link coupled linking group.
In further embodiment, accessory molecule at first with solid phase such as column material, as Sephadex or sepharose 4B, or the surface, covalently bound as reaction vessel.Then, polypeptide is loaded on the column material that is loaded with accessory molecule and by methods known in the art carries out coupling, as being undertaken by the method described in the chapters and sections of above " with ... coupling ".This method allows the polypeptide conjugate to separate from accessory molecule by eluting.The polypeptide conjugate carries out eluting by routine techniques under the physico chemical factor that does not cause the substantive degraded of polypeptide conjugate.The mobile phase that contains the polypeptide conjugate is separated and accessory molecule is still covalently bound from solid phase.Separation can otherwise be finished: for example, accessory molecule can pass through the molecule (as biotin) that specific-binding agent (as, streptavidin) discerns with second kind and derive.Therefore specific-binding agent can be connected with solid phase and allow by second accessory molecule of flowing through-solid phase column, and eluting accessory molecule-second molecular complex subsequently, but not the polypeptide conjugate can make the polypeptide conjugate separate with accessory molecule-second molecular complex.The polypeptide conjugate discharges from accessory molecule in any suitable manner.Can finish deprotection by the condition that provides, wherein accessory molecule separates from the functional site of its bonded FVII.For example, with link coupled antibody of polymer and anti-idiotype antibody between complex can be by pH regulator be separated to acidity or alkaline pH.More preferably use the conformation specific antibody of the special conformation of Ca2+ of identification FVII, under temperate condition, use the EDTA eluting at last.
The connection of serpin
The connection of serpin can be carried out according to method described in the WO96/12800.
The coupling of labeling polypeptide
In other embodiments, polypeptide is expressed as fusion rotein (with label, promptly usually by 1-30, the aminoacid sequence or the peptide section that constitute as 1-20 amino acid residue).Except allowing fast and easily carry out purification, label is to finish link coupled convenient tool between labeling polypeptide and the non-polypeptide fractions.Particularly, label is used on titer plate or other carrier such as the paramagnetic beads and finishes coupling, and the polypeptide of labelling can fix by label like this.Be that the polypeptide of labelling can directly be fixed on the titer plate (in principle without any purification) and carry out coupling from culture broth on titer plate with the link coupled advantage of the polypeptide of labelling.Therefore, can reduce the sum (from expressing coupling) of operating procedure.And label can play the work of spacerarm molecule in order to guarantee making fixed polypeptide be easier to coupling.The coupling that the usage flag polypeptide carries out can be the coupling with any non-polypeptide fractions disclosed herein, as with the coupling of polymer molecule such as PEG.
Identification to the particular marker used is not crucial, as long as this label can be expressed and can be fixed on the suitable surface or carrier mass with polypeptide.Many suitable labels can obtain by the commercial channel, for example, can purchase the Laboratories in Unizyme, Denmark.For example, described label can be arbitrary following sequence:
His-His-His-His-His-His
Met-Lys-His-His-His-His-His-His
Met-Lys-His-His-Ala-His-His-Gln-His-His
Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln
Or arbitrary following sequence:
EQKLI SEEDL (being described in Mol.Cell.Biol.5:3610-16,1985 C-end label)
DYKDDDDK (C-or N-end label)
YPYDVPDYA
The antibody of anti-above-mentioned label can obtain by the commercial channel usually, for example purchases in ADI AvesLab and Research Diagnostics.
The cut mark thing can be undertaken by commercially available enzyme from the polypeptide subsequently.
The method for preparing polypeptide of the present invention or conjugate polypeptide of the present invention
The polypeptide portion of polypeptide of the present invention or conjugate of the present invention (optional is the glycosylation form) can prepare by any suitable method known in the art.These methods comprise the nucleotide sequence that makes up this polypeptide of coding and express this sequence in suitable conversion or transfecting host.Preferred host cell is γ-carboxylated host cell, as mammalian cell.Yet, can produce polypeptide of the present invention by the combination of chemical synthesis process or chemical synthesis process or the combination of chemical synthesis process and recombinant DNA technology, but efficient is very low.
The nucleotide sequence of code book invention polypeptide or conjugate polypeptide portion can have the parental generation FVII of aminoacid sequence shown in the SEQ ID NO:1 by separation or composite coding, nucleotide sequence as hFVII makes up, and changes described nucleotide sequence then with introducing (promptly inserting or replacement) or elimination (promptly removing or replacement) related amino acid residue.
Nucleotide sequence can be modified through direct mutagenesis easily by known method.Perhaps, nucleotide sequence can prepare by chemosynthesis, for example, wherein come design oligonucleotides, and preferably select to help those codons of in the host cell of producing recombinant polypeptide, expressing based on required amino acid sequence of polypeptide by using oligonucleotide synthesizer.For example, can synthesize the micromolecule oligonucleotide of the required polypeptide each several part of a plurality of codings, by PCR, coupled reaction or ligase chain reaction (LCR) (Barany, PNAS 88:189-193,1991) described oligonucleotide be assembled up then.Each oligonucleotide contains 5 usually ' or 3 ' jag be used for complementary assembling.
Other nucleotide sequence method of modifying can be used for producing polypeptide variants so that high flow capacity screening, the method of disclosed relevant homology exchange among the US5093257 for example, and the method for related gene reorganization, be the reorganization between two or more homologous nucleotide sequences, cause producing and compare the new nucleotide sequence that has many nucleotide to change with the nuclei originis nucleotide sequence.Gene reorganization (being also referred to as DNA reorganization) relates to the random fragmentation of one or many nucleotide sequence and the circulation of ressembling, and selects coding to have the nucleotide sequence of the polypeptide of desired characteristic by screening thereupon.Reorganization can take place based on the nucleic acid of homology in order to make, and the relevant portion of described nucleotide sequence preferably has 50% homogeneity at least, the homogeneity as at least 60%, more preferably at least 70% homogeneity, the homogeneity as at least 80%.Reorganization can be carried out in external or body.
Suitable outer-gene reorganization method: Stemmer etc. have been shown in the following document, (1994), Proc.Natl.Acad.Sci.USA; Vol.91, pp.10747-10751; Stemmer (1994), Naturem vol.370, pp.389-391; Smith (1994), Nature vol.370, pp.324-325; Zhao etc., Nat.Biotechnol.1998.Mar; 16 (3): 258-61; Zhao H. and Arnold, FB, Nucleic AcidsResearch, 1997, Vol.25.No.6 pp.1307-1308; Shao etc., Nucleic Acids Research1998, Jan 15; 26 (2): pp.681-83; And WO95/17413.
Reorganization method in a kind of suitable body is disclosed among the WO97/07205.Other technology of carrying out nucleic acid mutation by reorganization in external or the body exist, as open among WO97/20078 and the US5837458.Special shuffling technology comprises " gene cluster reorganization (Family shuffling) ", " synthetic reorganization " " computerization (insliico) reorganization ".
Gene cluster reorganization is to make the gang's homologous genes from different genera carry out the reorganization of one or many and the circulation of follow-up screening or selection.The gene cluster shuffling technology has explanation in some documents, as: Crameri etc., (1998), Nature, vol.391, pp.288-291; Christians etc., (1999), NatureBiotechnology, vol.17, pp.259-264; Chang etc. (1999), Nature Biotechnology, vol.17, pp.793-797; And Ness etc. (1999), Nature Biotechnology, vol.17,893-896.
Synthetic reorganization relates to provides eclipsed synthetic oligonucleotide library, this library based on, as the sequence alignment between the purpose homologous genes.This synthetic oligonucleotide is recombinated, and the recombinant nucleic acid sequence that screening obtains can further be reorganized circulation as needs.Among the WO00/42561 relevant for the explanation of synthetic reorganization.
Computerization (In silico) reorganization refers to a kind of DNA reorganization process, is undertaken or simulate by computer system, so partially or completely need not carry out physical operations to nucleic acid.The explanation of reorganizing relevant for computerization (in silico) among the WO00/42560.
In case after the assembling (by synthetic, direct mutagenesis or other method), the nucleotide sequence of coded polypeptide is inserted in the recombinant vector immediately, and in the target transformed host cell, expresses essential regulating and controlling sequence with FVII and be operably connected.
Certainly, should understand the nucleotide sequence that not all carrier and expression regulation sequence can both be expressed polypeptide variants described herein equally well.Not all host can both make the identical same good function of expression system performance.Yet those skilled in the art need not experimentize and just can make one's options to these carriers, expression regulation sequence and host.For example, when selecting carrier, must consider the host, can be incorporated in the chromosome because carrier must duplicate maybe therein.Also should consider ability and any other protein of vector encoded such as the expression of antibiotic marker thing of carrier copy number, control copy number.When selecting expression regulation sequence, also should consider various factors.These factors comprise relative length as sequence, its controllability and with the compatibility of the nucleotide sequence of coded polypeptide, to consider potential secondary structure especially.Should consider itself and the toxicity of the compatibility of selected carrier, nucleotide sequence coded product, host's secretion characteristic and host correctly ability, host's fermentation and the difficulty or ease of cultivating requirement and nucleotide sequence coded product purification of folding polypeptide when selecting the host.
Recombinant vector can be the carrier of self-replicating, and promptly carrier exists as the outer entity of chromosome, and it duplicates and is independent of Chromosomal duplication, for example plasmid.In addition, when introducing host cell, carrier can be incorporated in the host cell gene group and duplicate with the chromosome of its integration.
Carrier is expression vector preferably, and wherein the nucleotide sequence of code book invention polypeptide can be transcribed required other segment with nucleotide sequence and is operably connected.Carrier is derived by plasmid or viral DNA usually.The various suitable expression vector that are used for expressing at host cell described herein are commercially available or document description are arranged.The expression vector that can be used for eucaryon host comprises as containing the carrier from the expression regulation sequence of SV40, bovine papilloma virus, adenovirus and cytomegalovirus.Concrete carrier such as pCDNA3.1 (+) Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, LaJola, CA, USA).The expression vector that can be used for yeast cells comprise 2 (plasmid and derivant thereof, (US 4,931 for the POT1 carrier, 373), the pJSO37 carrier (is described in Okkels, Ann.New York Acad.Sci.782,202-207,1996) and pPICZ A, B or C (Invitrogen).The expression vector that can be used for insect cell comprises pVL941, pBG311 (Cate etc., ＂ Isolation of Bovine and Human Genes forMullerian Inhibiting Substance And Expression of the Human Gene In AnimalCells ＂, Cell, 45, pp.685-98 (1986), pBluebac4.5 and pMelbac (all from Invitrogen).The expression vector that can be used for bacterial host comprises known bacterial plasmid, Tathagata comprises pBR322 from the plasmid of escherichia coli, and pET3a and pET12a are (from Novagen Inc., WI, USA), the plasmid of broad host range, as RP4, phage DNA, the various derivants of bacteriophage lambda for example, for example NM989 and other DNA phage are as M13 and thread single stranded DNA phage.
Other carrier that uses among the present invention comprises increase those carriers of a plurality of copies of the nucleotide sequence that allows coded polypeptide.This carrier that increases is well known in the art.For example, they comprise can be by DHFR amplification carrier (for example referring to Kaufman, U.S.Pat.No.4,470,46l, Kaufmanand Sharp, ＂ Construction Of A Modular Dihydrofolate Reductase cDNA Gene:Analysis Of Signals Utilized For Efficient Expression ＂, Mol.Cell.Biol., 2, pp.1304-19 (1982)) and can pass through glutamine synthetase (＂ GS ＂) amplification carrier (for example referring to US5,122,464 and EP338,841).
Recombinant vector can further comprise the DNA sequence that described carrier is duplicated in described host cell.An example (when host cell is mammalian cell) of this sequence is the replication origin of SV40.When host cell was yeast cells, the proper sequence that carrier is duplicated was yeast plasmid 2 (replicator REP1-3 and a replication origin.
Carrier also can contain selectable labelling, as gene, its product replenishes the defective of host cell, as the gene (P.R.Russell of dihydrofolate reductase of encoding (DHFR) or schizosaccharomyces pombe TPI, Gene40,1985, pp.125-130), perhaps give gene to the resistance of medicine such as ampicillin, kanamycin, tetracycline, chloromycetin, neomycin, hygromycin or methotrexate.For saccharomyces cerevisiae, selectable sign comprises ura3 and Leu2.For filamentous fungi, selectable labelling comprises AmdS, pyrG, arcB, NiaDWith SC
Term " regulating and controlling sequence " comprises in this article to the essential or favourable all the components of expression of polypeptides of the present invention.Each regulating and controlling sequence can be natural or external concerning the nucleotide sequence of coded polypeptide.These regulating and controlling sequences include but not limited to leader region, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activation sequences, signal peptide sequence and transcription terminator.Regulating and controlling sequence comprises promoter at least.
The present invention can use various expression regulation sequences.These spendable expression regulation sequences comprise the expression regulation sequence that links to each other with the structural gene of aforementioned expression vector and any sequence and the various combination thereof of known regulation and control protokaryon or eukaryotic cell or its viral gene expression.
Early stage or the late promoter that comprises SV40 and adenovirus in the mammalian cell with the example of transcribing relevant suitable regulating and controlling sequence, major late promoter as adenovirus 2, MT-1 (metallothionein gene) promoter, human cytomegalic inclusion disease virus immediate early gene promoter (CMV), people's EF-1 ((EF-1 () promoter, the minimum heat shock protein 70 promoter of fruit bat, rous sarcoma virus (RSV) promoter, people's ubiquitin C (UbC) promoter, human growth hormone's terminator, SV40 or adenovirus E 1 b district's polyadenylation signal and Kozak consensus sequence (Kozak, M.J Mol Biol 1987 Aug 20; 196 (4): 947-50).
In order to improve the expression in mammalian cell, synthetic intron can be inserted 5 ' untranslated region of the nucleotide sequence of coding said polypeptide.The example of synthetic intron be from plasmid pCI-Neo synthetic intron (available from Promega Corporation, WI, USA).
Instruct the example of the suitable regulating and controlling sequence of transcribing to comprise polyhedrin promoter, P10 promoter, autographa california (Autographa californica) polyhedrosis virus basic protein promoter, baculovirus immediate early gene 1 promoter and baculovirus 39K delayed early gene promoter and SV40 polyadenylation sequence in the insect cell.The example of the suitable regulating and controlling sequence that uses in yeast host cell comprises yeast (promoter of mating system, yeast triose-phosphate isomerase (TPI) promoter, the promoter from Yeast sugar zymolysis gene or alcohol dehydrogenase gene, ADH2-4c promoter and induction type GAL promoter.The example of the suitable regulating and controlling sequence that uses in filamentous fungal host cell comprises ADH3 promoter and terminator, by coding aspergillus oryzae TAKA amylase triose-phosphate isomerase or alkaline protease, aspergillus niger (the deutero-promoter of gene, TPI1 terminator and the ADH3 terminator of amylase, aspergillus niger or aspergillus nidulans (A.Nidulans) glucoamylase, aspergillus nidulans acetamidase, Rhizomucor miehei (Rhizomucor miehei) aspartic protease or lipase.The example of the suitable regulating and controlling sequence that uses in bacterial host cell comprises the promoter and (the main promoter region of phage of 1ac system, trp system, TAC or TRC system.
The existence of signal peptide or do not exist as depend on the expression host cell that is used for polypeptide and produces, by expressed protein (being intracellular or extracellular protein) with whether need to secrete.In order to use in filamentous fungi, signal peptide can conveniently be derived by the gene of gene, coding Palatase or the protease or the Humicola lanuginosa lipase of the amylase of coding aspergillus bacterial strain or glucoamylase.Signal peptide preferably by coding aspergillus oryzae TAKA amylase, aspergillus niger neutrality (derive by the gene of amylase, aspergillus niger acid-stable starch enzyme or aspergillus niger glucoamylase.In order in insect cell, to use, signal peptide can derive easily by insect genes (referring to WO90/05783), as (egt) (Murphy etc. of Lepidoptera Manduca sexta adipokinetic hormone precursor (US5023328), bee variety toxin (Invitrogen), ecdysteroids UDP glucosyltransferase (glucosyltransferase), ProteinExpression and Purification 4,349-357 (1993)) or people's pancreatic lipase (hpl) (Mefhods inEnzymology 284, pp.262-272,1997).The preferred signals peptide that uses in the mammalian cell is hFVII's or Mus Ig (light chain signal peptide (Coloma, M (1992) J.Imm.Methods 152:89-104).In order in yeast cells, to use, find that the appropriate signal peptide is that (the carboxypeptidase signal peptide of factor signal peptide (referring to US4870008), modification is (referring to people such as L.A.Valls for saccharomyces cerevisiae, Cell 48,1987, pp.887-897), yeast BAR1 signal peptide (referring to WO87/02670), yeast aspartic protease 3 (YAP3) signal peptide are (referring to people such as M.Egel-Mitani, Yeast 6,1990, pp.127-137) and synthetic targeting sequencing TA57 (WO98/32867).Colibacillary appropriate signals peptide is signal peptide ompA (EP581821).
The encode nucleotide sequence of FVII polypeptide of the present invention no matter by direct mutagenesis, synthetic, PCR or other which kind of method preparations, selectively comprises the nucleotide sequence of coded signal peptide.Need from the cell of expressing, secretion then need signal peptide as polypeptide.The sort signal peptide if exist, should be the cell recognition that can be expressed described polypeptide.Signal peptide can with described homologous peptide (as, usually link to each other with hFVII) or allos (differing from hFVII as its origin), perhaps can with host cell homology or allos, promptly common described signal peptide is the signal peptide of this host cell expression or it is not the signal peptide of this host cell expression usually.Accordingly, described signal peptide can be a protokaryon, as derives from antibacterial such as escherichia coli, or eucaryon, as derive from mammal or insecticide or yeast cells.
Can use any suitable hosts to produce the polypeptide of conjugate of the present invention, comprise antibacterial, fungus (comprising yeast), plant, insecticide, mammal or other suitable zooblast or cell line and transgenic animal or plant.The example of bacterial host cell comprises gram-positive bacterium such as bacillus, as bacillus brevis or bacillus subtilis, Pseudomonas or streptomyces or gram negative bacteria, as coli strain.Carrier can be introduced bacterial host cell, for example can pass through protoplast transformation (referring to as Chang and Cohen, 1979, Molecular General Genetics 168:111-115), use experience attitude cell is (referring to as YOung and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology56:209-221), electroporation is (referring to as Shigekawa and Dower, 1988, Biotechniques6:742-751) or coupling (referring to as Koehler and Thorne, 1987, Journal of Bacteriology169:5771-5278) carry out.The example of suitable filamentous fungal host cell comprises the aspergillus bacterial strain, as aspergillus oryzae, aspergillus niger or aspergillus nidulans, and Fusarium or trichoderma.The fungal cell can transform, and conversion process relates to protoplast formation, protoplast transformation and cell wall and regenerates by known mode own.The appropriate method that transforms the aspergillus host cell has been described in EP238023 and US5679543.People such as Malardier 1989, the appropriate method that transforms Fusarium has been described among Gene 78:147-156 and the WO96/00787.The example of suitable yeast host cell comprises the bacterial strain of saccharomyces, as saccharomyces cerevisiae genus, Schizosaccharomyces, Kluyveromyces, pichia, as pichia pastoris phaff or P.Methanolica, Hansenula, as multiple-shaped nuohan inferior yeast or Yarrowia.Yeast can use Becker and Guarente, In Abelson, J.N.And Simon, M.I., editors, Guide to Yeast Geneticsand Molecular Biology, Methods in Enzymology Volume 194, pp 182-187, Academic Press, Inc., New York; People such as Ito, 1983, Journal of Bacteriology 153:163; With people 1978 such as Hinnen, Proceedings of the National Academy of Sciences USA75:1920; And by Clontech Laboratories, Inc, Palo Alto, CA, the method described in the USA (product manual of YeastmakerTM yeast conversion system test kit) transforms.The example of suitable insect host cell comprises Lepidoptera cell line, as fall army worm (Sf9 or Sf21) or cabbage looper cell (High Five) (US5077214).Can transform insect cell and produce heterologous polypeptide therein by the described method of Invitrogen.The example of suitable mammalian host cell comprises that Chinese hamster ovary (CHO) cell line is (as, CHO-K1; ATCC CCL-61), grivet cell line (COS) (as, COS1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); Mouse cell (as, NS/O), hamster children Ren Mus dirty (BHK) cell line (as, ATCC CRL-1632 or ATCC CCL-10) and people's cell (as, HEK293 (ATCC CRL-1573)), and the plant cell in the tissue culture.Suitable in addition cell line is well known in the art and can be available from common preservation center such as American type culture collection, Rockville, Maryland.And mammalian cell (as Chinese hamster ovary celI) can carry out modification by method as described in the US5047335 and express sialyltransferase, as, 1, the 6-sialyltransferase is so that improve the glycosylation of FVII or FVIIa polypeptide.
In order to increase secretion, make polypeptide of the present invention and endo protease, PACE (paired basic amino acid invertase) (described in US5986079) especially, synthetic together as Kex2 endo protease (described in WO00/28065) is favourable.
The method that exogenous DNA is introduced mammalian host cell comprises the transfection of transfection, electroporation, the mediation of DEAE-glucosan of calcium phosphate mediation, liposome-mediated transfection, viral vector and by LifeTechnologies Ltd, Paisley, the transfection method of the described use of UK Lipofectamin2000.These methods are well known in the art, for example at people such as Ausbel (eds.), and 1996, CurrentProtocols in Molecular Biology, John Wiley ﹠amp; Sons, New York, described in the USA.Carry out the cultivation of mammalian cell by the method for setting up, for example at Animal Cell Biotechnology, Methods and Protocols, Nigel Jenkins compiles, and 1999, Human Press Inc, Totowa, NewJersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, disclosed among the Cambridge University Press 1997.
In production method of the present invention, with methods known in the art cultured cell in being suitable for producing the Nutrient medium of polypeptide.For example, cell can be in laboratory by shake-flask culture, small-scale or large scale fermentation (comprise continuously, in batch, fed-batch or solid state fermentation) or at suitable culture medium with allow expression and/or separate under the condition of described polypeptide and carry out industrial fermentation.Cultivation uses methods known in the art to carry out in the suitable nutrient medium that contains carbon and nitrogenous source and inorganic salt.Proper culture medium is commercially available maybe can be prepared by disclosed composition (for example, described in the catalogue of American type culture collection).If the polypeptide secretion is in Nutrient medium, polypeptide can directly reclaim from culture medium.If polypeptide is not secreted, can from cell lysate, reclaim.
Can reclaim the gained polypeptide by methods known in the art.For example, can include but not limited to that centrifugal, filtration, extraction, spray drying, evaporation or precipitation reclaim polypeptide from Nutrient medium by conventional method.
Can by the whole bag of tricks known in the art include but not limited to chromatography (as, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (as, the isoelectrofocusing of preparation property), dissolubility difference (as, ammonium sulfate precipitation), SDS-PAGE or extraction (referring to, as Protein Purification, J.-C.Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) come purified polypeptide.
Disclose purification of single stranded FVII in the literature and its activation has been the several different methods of double-stranded FVIIa (Broze and Majerus, 1980, J.Biol.Chem.255:1242-47, Hedner and Kisiel, 1983, J.Clin.Invest.71:1836-41 etc.).A kind of method that can be used for purification of single stranded FVII is to introduce zinc ion as described in US5700914 in purge process.
In the preferred embodiment, described polypeptide is purified as strand FVII, and further by PEGization.The FVII single chain polypeptide activation of PEGization is by use immobilized enzyme (as factor IIa, IXa, Xa and XIIa), or by using cation exchange substrate or its analog self-activation.
At first the FVII of purification of single stranded form carries out PEGization (if desired) then, is favourable by one of said method or as (1989, Biochemistry 28:9331-36) such as Pedersen described self-activation activates at last.The advantage of carrying out PEGization before activation is to have avoided to the formed new aminoterminal PEGization of the shearing of R152-I153.New aminoterminal PEGization can make described molecular inactivation, because the hydrogen bond that forms between D242 and the I153 amino terminal is active necessary.
Medical composition and its use of the present invention
Another aspect the present invention relates to compositions, particularly a kind of pharmaceutical composition, and it comprises polypeptide of the present invention or conjugate (conjugate that comprises above-mentioned non-activity) and pharmaceutically suitable carrier or excipient.
Conjugate of the present invention, polypeptide or pharmaceutical composition can be used as medicine.
Preferably, described polypeptide or described (activity) conjugate can be used for FVIIa/TF relevant disease or disorderly medicine in production for treating or the prevention mammal.For example described polypeptide or described (activity) conjugate can be used for production for treating or prophylactic medicine, and wherein accelerating blood coagulation is favourable for described disease, the patient of the not enough disease of blood coagulation when suffering from blood vessel injury as treatment.Especially, described polypeptide or described (activity) conjugate can be used as the medicine of the following disease of production for treating, hemophilia, hemophilia with FVIII and FIX inhibitor, thrombocytopenic patient, thrombocytopathic patient, as Glan now Man thrombasthenia platelet discharge defective and depots defective, the patient who suffers from von Willebrand disease, the patient who suffers from hepatic disease, or the healthy person of severe haemorrhage problem arranged, as having produced inhibitor because of wound or major operation at FVIIa, hemorrhage disorder, as hemophilia and other usually with serious tissue injury diseases associated.
Similarly, non-activity conjugate of the present invention can be used as production for treating or prevention mammal FVIIa/TF relevant disease or disorderly medicine.For example, non-activity conjugate of the present invention can be used as production for treating or prophylactic medicine, wherein reducing blood coagulation is favourable for described disease, be in the highly patient of blood coagulation state as prevention or treatment, as sepsis, the venothrombotic patient of the degree of depth, easily suffer from myocardial infarction or thrombosis apoplexy, the patient of lung infraction, the patient of acute coronary syndrome (acute coronarysyndromes) (myocardial infarction and unsettled angina pectoris), coronary heart disease (coronarycardiac) patient, the heart disease and the restonosis of blood vessel molding patient with operation accepted in prevention, and the patient who suffers from peripheral blood vessel.Non-activity conjugate of the present invention also can be used for the medicine of production for treating respiratory disorder, tumor growth and transfer.
On the other hand, described polypeptide, described (activity) conjugate or the described pharmaceutical composition that comprises the active conjugate of the present invention can be used for treatment and suffer from the mammiferous method of FVIIa/TF relevant disease or disorder (as one or more the above disease or disorder), comprise that the mammal that needs treatment is with this peptide species, conjugate or the compositions of effective dose.
Similarly, described non-activity conjugate or the described pharmaceutical composition that comprises non-activity conjugate of the present invention can be used for treatment to be suffered from the mammiferous method of FVIIa/TF relevant disease or disorder (as one or more the above disease or disorder), and comprising needs the mammal treated this non-activity conjugate or the compositions with effective dose.
Give polypeptide of the present invention or conjugate with the treatment effective dose, described dosage approximately with rFVII as
Figure C01804864D0049100412QIETU
The dosage that treatment is adopted is identical or higher." treatment effective dose " is meant the dosage that controlling morbid state is enough to produce Expected Results herein.The accurate dosage of administration depends on concrete condition, can be determined by known method by those skilled in the art.In general, described dosage should prevent from or reduce to be treated the seriousness or the diffusion of disease or symptom.The effective dose that it will be apparent for a person skilled in the art that polypeptide of the present invention, conjugate or compositions depend on disease, dosage, administration time table, polypeptide or conjugate or compositions be individually dosed or with the serum half life of other therapeutic agent administering drug combinations, compositions and patient's general health.Preferably, polypeptide of the present invention, conjugate or compositions give with effective dose, especially to be enough to that the dosage of coagulopathy normalization is given.
Polypeptide of the present invention or conjugate are preferably with the composition forms administration, comprising pharmaceutically suitable carrier or excipient." pharmaceutically acceptable " means carrier or the excipient that does not cause any detrimental effect in the patient of administration.These pharmaceutically suitable carrier and excipient be in the art known (referring to, as Remington ' sPharmaceutical Sciences, 18th edition, A.R.Gennaro, Ed., Mack PublishingCompany (1990); Pharmaceutical Formulation Development of Peptides andProteins, S.Frokjaer and L.Hovgaard, Eds., Taylor ﹠amp; Francis (2000); With Handbook of Pharmaceutical Excipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press (2000)).
Polypeptide of the present invention or conjugate can be mixed with pharmaceutical composition by known method.At E.W.Martin (Mark Publ company, 16 editions, 1980) Remington ' s Pharmaceutical Sciences appropriate formulation has been described.
Polypeptide of the present invention or conjugate can use and/or use with the form of its salt by original shape.Suitable salt includes but not limited to the salt with alkali metal or alkaline-earth metal such as sodium, potassium, calcium and magnesium formation, and as zinc salt.These salt or complex can be used as crystal and/or impalpable structure exists.
Pharmaceutical composition of the present invention can give separately also can unite with the other treatment preparation to give.The ingredient that these preparations can be used as in the same pharmaceutical composition gives, and perhaps gives respectively simultaneously or according to certain treatment time table with polypeptide of the present invention or conjugate.In addition, polypeptide of the present invention, conjugate or pharmaceutical composition can be used as the adjuvant of other treatment.
" patient " comprises people and other mammals among the present invention.Therefore described method is medical for animalsly all can.The pharmaceutical composition of polypeptide of the present invention or conjugate can dispose in a variety of forms, as with liquid, and gel, the solid form of lyophilized powder or compacting.And different, this is conspicuous to preferred form for those skilled in the art according to the special needs for the treatment of.
Especially, the pharmaceutical composition of polypeptide of the present invention or conjugate is with lyophilized powder or the preparation of stabilizing solution form.Described polypeptide or conjugate can be by multiple known method lyophilizing efflorescence.Polypeptide or conjugate can be prepared into stable solution form by the proteolysis site is removed or covered.The advantage that the obtains stabilizing solution patient that is to be more convenient for uses, and when emergency, act on faster, this possible can lifesaving.And different, this is tangible to preferred form for those skilled in the art according to the specific needs for the treatment of.
Preparation of the present invention administration in many ways includes but not limited to, in oral, subcutaneous, intravenous, the cerebellum, in the intranasal, intradermal, intraperitoneal, intramuscular, lung, transvaginal, per rectum, ophthalmic or in any other suitable mode.But the preparation continous pouring gives, although bolus injection (bolusinjection) is acceptable, need utilize techniques well known, as pump or implantation.Under the certain situation described preparation can solution or spraying directly give.
Parenteral administration agent (Parentals)
The preferred embodiment of pharmaceutical composition is the solution that is designed to parenterai administration.Although under many circumstances, pharmaceutical solution formulations can provide to be applicable to the liquid form that uses immediately, and described parenteral formulation also can provide with freezing or lyophilized form.Under the former situation, described compositions must be thawed before use.The stability of active component under various storage requirements that back one dosage form is generally used in the enhancing composition being comprised, as well known by persons skilled in the art, lyophilized formulations is more stable than its liquid homologue usually.Described lyophilized formulations is prepared again by adding one or more suitable pharmaceutically acceptable diluents such as sterilized water for injection or sterile saline solution before use.
Under the situation of parenteral administration, make lyophilized formulations or aqueous solution is standby, as preparing excipient such as buffer agent, stabilizing agent, antiseptic, isotonic agent, non-ionic surface active agent or detergent, antioxidant and/or other various additives by polypeptide that will have required purity and suitable mixing of pharmaceutically suitable carrier, excipient or stabilizing agent (being referred to as " excipient ") that one or more this areas are used always.
Buffer agent helps pH is remained in the scope near physiological condition.They exist with the concentration range of about 2mM-50mM usually.The suitable buffer agent that the present invention uses comprises organic and mineral acid and salt thereof such as citrate buffer agent (as, sodium dihydrogen citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-sodium dihydrogen citrate mixture etc.), the succinate buffer agent (as, succinic acid-succinic acid one sodium mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), the tartrate buffer agent (as, tartaric acid-sodium tartrate mixture, tartaric acid-Soluble tartar. mixture, tartaric acid-sodium hydroxide mixture etc.), the fumarate buffer agent (as, fumaric acid-fumaric acid one sodium mixture, fumaric acid-Disodium fumarate. mixture, fumaric acid one sodium-Disodium fumarate. mixture etc.), gluconate (gluconate) buffer agent (as, gluconic acid-gluconic acid sodium salt mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture etc.), the oxalates buffer agent (as, oxalic acid-Disodium oxalate. mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffer agent (as, lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffer (as, acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture etc.).Other possible buffer agent is phosphate buffer, histidine buffer and front three amine salt such as Tris.
Stabilizing agent relates to a big class excipient, its envelop of function from extender to the dissolution treatment agent or help to prevent degeneration or with the adherent additive of chamber wall.Common stabilizing agent can be alditol (above enumerating); Aminoacid such as arginine, lysine, glycine, glutamine, agedoite, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc., organic sugar or sugar alcohol, as lactose, trehalose, stachyose, mannitol, Sorbitol, xylitol, ribitol, inositol, galactitol, glycerol etc., comprise cyclic alcohol such as cyclohexanhexanol; Polyethylene Glycol; Amino acid polymer; The sulfur-bearing Reducing agent, as urea, glutathion, thioctic acid, sodium thioglycolate, thioglycerol, (single thioglycerol and sodium thiosulfate; Low molecular weight polypeptide (residue promptly＜10); Protein such as human serum albumin, bovine serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Monosaccharide such as xylose, mannose, fructose and glucose; Disaccharide such as lactose, maltose and sucrose; Trisaccharide such as Raffinose and polysaccharide such as glucosan.Calculate based on reactive protein weight, the scope that exists that stabilizing agent is common is the 0.1-10000 weight portion.
Add antiseptic and block growth of microorganism, the amount of adding is about 0.2%-1% (w/v) usually.The suitable preservatives that the present invention uses comprises phenol, benzyl alcohol, metacresol, oxybenzene formic acid (paraben) methyl ester, propylparaben, octadecyl dimethyl benzene ammonio methacrylate, benzylidene halogenide (as benzylidene chloride, bromide or iodide), chlorination hexane diamine, oxybenzene alkyl formate such as methyl ester or propyl ester, catechol, resorcinol, Hexalin and 3-amylalcohol.
Can add isotonic agent and guarantee the isotonicity of fluid composition, isotonic agent comprises alditol, and preferred three hydroxyls or more senior sugar alcohol are as glycerol, erithritol, 1,2,3,4,5-pentanepentol, xylitol, Sorbitol and mannitol.The amount of polyhydroxy-alcohol can be the 0.1%-25% weight ratio, is generally 1%-5%, calculates with the relative quantity of other component.
Can exist non-ionic surface active agent or cleaning agent (being also referred to as " wetting agent ") to help dissolution treatment agent and protection therapeutical peptide to avoid stirring inductive gathering, it also allows formula agent to be exposed to shear surface pressure and does not cause the polypeptide degeneration.Suitable ionic surfactant pack is drawn together polysorbate (polysorbate) (20,80 etc.), polyoxamer (184,188 etc.), Pluronic (polyhydric alcohol, polyoxyethylene sorbitan monoether (polysorbas20, Tween 80 etc.).
Other various excipient comprise extender or filler (as starch), chelating agen (as EDTA), antioxidant (as ascorbic acid, methionine, vitamin E) and cosolvent.
Active component also can be wrapped in the microcapsule of preparation, for example prepare by the coascervation technology or by interfacial polymerization, for example hydroxy methocel, gelatin or poly-(methyl first acrylate) microcapsule are wrapped in the colloidal drug delivery system (for example liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule) or are wrapped in the big Emulsion.These technology are disclosed in Remington ' s Pharmaceutical Sciences (the same).
The employed non-gastrointestinal preparation of vivo medicine-feeding must be sterilized.This process is finished easily, for example, filters by aseptic filter membrane.
Extended release preparation
The suitable example of extended release preparation comprises the solid hydrophobic polymer semipermeable materials that contains polypeptide or conjugate, the material with suitable form such as film or microcapsule.The example that continues releasable material comprises polyester, hydrogel (for example, poly-(2-hydroxyethyl first acrylate) or poly-(vinyl alcohol)), polyactide, L-glutamic acid and the copolymer of L-ethyl glutamate, nondegradable ethylene-ethyl acetate, degradable lactic acid-ethanol copolymer such as ProLease (technology or Lupron Depot ((Injectable microspheres that is made of lactic acid-ethanol copolymer and leuprorelin acetate) and gather-D-(-)-3-hydroxybutyric acid.Polymer such as ethylene-ethyl acetate and lactic acid-ethanol can discharge molecule for a long time as reaching or surpass 100 days, and some hydrogels discharges protein within a short period of time.When the polypeptide in the encapsulation kept in vivo for a long time, they caused loss of bioactivity and may change immunogenicity because of expose degeneration or gathering in the environment of 37 ℃ of humidities.Reasonably stable strategy can be according to related mechanism design.For example, if find to assemble mechanism is to exchange mutually by sulfo-disulphide to form intramolecularly S-S key, can reach stable by modification sulfydryl, lyophilizing acid solution, controlled humidity, suitable introducing agent and the exploitation specificity polymer-type compositions of use so.
In the following example, further describe the present invention, these embodiment and indefinite.
Sequence
SEQ ID NO:1 shows hFVII albumen (comprising γ-carboxylated residue)
The proteic cDNA sequence of SEQ ID NO:2 code displaying hFVII
SEQ ID NO:3 shows hFVII albumen (no γ-carboxylated residue)
SEQ ID NO:4 shows the expression cassette that FVII albumen is expressed in mammal
SEQ ID NO:5 shows the CBProFpr174 primer
SEQ ID NO:6 shows the CBProFpr175 primer
SEQ ID NO:7 shows the CBProFpr216 primer
SEQ ID NO:8 shows the CBProFpr229 primer
SEQ ID NO:9 shows the CBProFpr221 primer
SEQ ID NO:10 shows the CBProFpr228 primer
SEQ ID NO:11 shows the CBProFpr226 primer
Material and method
Be used for definite amino acid whose method that will change
Come-at-able surf zone (ASA)
The come-at-able surf zone (ASA) of each atom in the program groups that uses a computer (B.Lee and F.M.Richards, J.Mol.Biol.55:379-400 (1971)) version 2 (Copyright (c) the 1983 Yale University) computation structure.This method uses 1.4 usually
Figure C01804864D0053100647QIETU
The probe of size also is defined as the area that probe core forms with come-at-able surf zone (ASA).Before this calculates, from coordinate, remove all hydrones and all hydrogen atoms, and other atom that does not directly link to each other with this protein.
The classification of side chain (fractional) ASA
The classification ASA of side chain atom calculates divided by the ASA representative value of remaining type side chain atom in the ALA-x-ALA tripeptides that prolongs by the ASA summation of atom in the side chain.Referring to Hubbard, Campbell ﹠amp; Thornton (1991) J.Mol.Biol.220,507-530.In this embodiment, the CA atom is counted as the part of glycine residue side chain but is not counted as the part of other residue.Following table is represented the 100%ASA standard of side chain:
Undiscovered residue is accredited as and has 100% exposure on this structure, because of it is considered to be positioned at Hookean region.Position 6,7,14,16,19,20,25,26,29 and the Gla at 35 places all be confirmed as 100% and expose.
Determine the distance between the atom
Utilize the molecule mapping software, (v.98.0 MSI INC can determine the distance between the atom easily as Insight II.
The catalytic site district
The catalytic site area definition is any such residue, described residue have at least one atom the catalysis triplet (residue H193, D242, S344) in any atom In the distance.
Determine the tissue factor binding site
Receptor binding site is defined as comprising all these class residues, and described residue is when receptors bind, and its come-at-able surf zone changes.This calculates to determine by at least two ASA; One based on isolating aglucon in aglucon/receptor complex, and another is based on complete aglucon/receptor complex.
Detect the method for FVII and FVIIa character
The method of half life in the measuring ability gonosome
The detection of biological halflife can be undertaken by the several different methods that illustrates in the above-mentioned document in the body.The method of half life in the body that detects rFVIIa or its variant has been described among the FDA reference number 96-0597.Briefly, give described conjugate, extracting blood plasma before polypeptide or the compositions and in 24 hours afterwards, detecting FVII blood coagulation activity in this blood plasma.Detect the intermediate value of volume of distribution under the steady statue, and definite intermediate value clearance rate.
Detection to the reduction of proteolysis sensitivity
The preparation compositions, described compositions comprises conjugate (100-750 μ g/ml, preferred 600 μ g/ml), 1.5mg Ca2+/ml (with the calcium chloride form), mannitol (30mg/ml), polysorbate 80 (0.1mg/ml), sodium chloride (3mg/ml), and glycyl-glycine buffer (1.3mg/ml, pH5.5).
Preparation contains the analogous composition of wild type rFVIIa.
With method described in " detecting the method for blood coagulation activity " or " detecting the method for low-level catalytic activity " or " detecting the method for catalytic activity " chapters and sections, determine initial blood coagulation activity, or initial amide decomposition activity.
It is active at least 25% then compositions to be lost its initial blood coagulation or amidatioon at 37 ℃ of incubations up to the compositions that contains wild type rFVIIa, and preferably at least 50%.
Detect the blood coagulation or the amide decomposition activity of the compositions that comprises conjugate of the present invention then.
Represent that with percentage ratio conjugate of the present invention compares with wild type rFVIIa, to the reduction of the sensitivity of proteolysis.
Other detection methods of the sensitivity that proteolysis is reduced
Proteolysis can detect by the method for explanation among the embodiment 5 of US5580560, and wherein said Proteolytic enzyme is an autoproteolytic cleavage.
In addition, the proteolysis of reduction can utilize the radioactive label sample to detect in the model in vivo, by blood sampling, and these blood samples is carried out the proteolysis effect that SDS-PAGE and autoradiography contrast wild type and conjugate.
Which kind of no matter uses analyze determine proteolysis, " proteolysis of reduction " is meant the obvious reduction of comparing with non-link coupled wild type FVIIa on the cracking degree, this is to measure by gel scanning, HPLC to the SDS-PAGE gel of coomassie brilliant blue staining, or utilizes following colour test to determine by the conservative catalytic activity with respect to wild type.
To determining of the molecular weight of rFVII and conjugate thereof
The molecular weight of link coupled or non-link coupled rFVII or its conjugate is by SDS-PAGE, gel filtration, Western trace, the auxiliary laser desorption of substrate (matrix assisted laser deserption), mass spectral analysis or equilibrium centrifugation, as according to Laemmli, (Nature Vol 227 (1970) for UK, pp.680-85) SDS-PAGE determines.
Detect the method for low-level catalytic activity
With FVII (Chromogenix, Art.No 82 19 00) determines the amide decomposition activity in the culture fluid that FVII/FVIIa dilute sample and fermentation liquid/process cultivates.Operation instruction according to the manufacturer is measured amide decomposition activity.Concise and to the point, FX exists with excessive, and is changed into FXa at 37 ℃ by FVIIa.(N-α-Cbo-D-Arg-Gly-Arg-pNA), discharge colour developing molecule p-nitro-benzene amine (para-nitro-anillin) (pNA), absorbing wavelength is the light of 405nm to the FXa hydrolysis chromogenic substrate S2765 that is obtained.Add the acetic acid cessation reaction.By with the comparison of the standard curve of FVIIa (test buffer in 125pg/ml-1ng/ml), determine the amount of FVII/FVIIa in the sample.
Detect the method for catalytic activity
The ability of the little peptide substrates of conjugate cracking can detect by using chromogenic substrate S-2288 (D-Ile-Pro-Arg-p-Nitraniline .).Reorganization FVIIa is diluted in the 0.1M Tris that contains 0.1%BSA, 0.1M NaCl, 5mM CaCl 2, among the pH8.3.Add S-2288 to 1mM starting reaction, 37 ℃ of incubations detect the absorbance of 405nm after 30 minutes.
Detect the method for blood coagulation activity
Use WO92/15686 (one-stage) coagulation analysis of described one step of standard to detect the activity of FVIIa.In brief, testing sample dilutes among the 0.1%BSA at 50mM Tris (pH7.5), gets the Thromboplastin C incubation that this solution 100 μ l and 100 μ l FVII deficiency blood plasma and 200 μ l contain 10mM Ca++.Detect clotting time and compare the human normal plasma's of described standard curve use Citrated serial dilutions preparation with standard curve.
Detect the method for anticoagulant active
The anticoagulant active of non-activity FVII or FVIIa conjugate can detect by an above-mentioned step coagulation analysis (detecting the method for blood coagulation activity), wherein the relipidated tissue factor of non-activity conjugate and wild type FVII competition limited quantity.Analyze substantially according to WO92/15686, method described in the EXAMPLE III is carried out, and the text is incorporated herein by reference.The ability that record non-activity conjugate prolongs wild type FVII clotting time is with its index as anticoagulant active.
Embodiment
Embodiment 1
Present embodiment uses (J Mol Biol, 1996 such as Banner; 285:2089) definite and soluble tissue factor form the x-ray structure of the hFVIIa of complex.Notice that the residue sequence number in the reference literature is not consistent with this sequence.Sequence number used herein is consistent with SEQ ID NO:1.Position 6,7,14,16,19,20,25,26,29 and the Gla at 35 places at this equal called after GLU (triliteral writing a Chinese character in simplified form) or E (single-letter is write a Chinese character in simplified form).The residue that does not have the 143-152 position in the described structure.
The surface exposes
The classification ASA that the FVII fragment is carried out separately calculates, in conjunction with in the said method to non-standard and/or lose the definition of the accessibility of residue, determine that following residue has the side chain above 25% to be exposed to its surface: A1, N2, A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, E16, K18, E19, E20, Q21, S23, F24, E25, E26, R28, E29, F31, K32, D33, A34, E35, R36, K38, L39, W41, I42, S43, S45, G47, D48, Q49, A51, S52, S53, Q56, G58, S60, K62, D63, Q64, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82, T83, H84, K85, D86, D87, Q88, L89, I90, V92, N93, E94, G97, E99, S103, D104, H105, T106, G107, T108, K109, S111, R113, E116, G117, S119, L120, L121, A122, D123, G124, V125, S126, T128, P129, T130, V131, E132, I140, L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157, V158, P160, K161, E163, L171, N173, G174, A175, N184, T185, I186, H193, K197, K199, N200, R202, N203, I205, S214, E215, H216, D217, G218, D219, S222, R224, S232, T233, V235, P236, G237, T238, T239, N240, H249, Q250, P251, V253, T255, D256, E265, R266, T267, E270, R271, F275, V276, R277, F278, L280, L287, L288, D289, R290, G291, A292, T293, L295, E296, N301, M306, T307, Q308, D309, L311, Q312, Q313, R315, K316, V317, G318, D319, S320, P321, N322, T324, E325, Y326, Y332, S333, D334, S336, K337, K341, G342, H351, R353, G354, Q366, G367, T370, V371, G372, R379, E385, Q388, K389, R392, S393, E394, P395, R396, P397, G398, V399, L400, L401, R402, P404 and P406.
Following residue has the side chain above 50% to be exposed to the surface:
A1, A3, F4, L5, E6, E7, L8, R9, P10, E14, E16, K18, E19, E20, Q21, S23, E25, E26, E29, K32, A34, E35, R36, K38, L39, I42, S43, G47, D48, A51, S52, S53, Q56, G58, S60, K62, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82, H84, K85, D86, D87, Q88, L89, I90, V92, N93, E94, G97, T106, G107, T108, K109, S111, E116, S119, L121, A122, D123, G124, V131, E132, L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157, P160, N173, G174, A175, K197, K199, N200, R202, S214, E215, H216, G218, R224, V235, P236, G237, T238, H249, Q250, V253, D256, T267, F275, R277, F278, L288, D289, R290, G291, A292, T293, L295, N301, M306, Q308, D309, L311, Q312, Q313, R315, K316, G318, D319, N322, E325, D334, K341, G354, G367, V371, E385, K389, R392, E394, R396, P397, G398, R402, P404 and P406.
The tissue factor binding site
Calculate by ASA, the following residue among the people FVII changes its ASA in complex.These residues are defined as forming the residue of receptor binding site: L13, K18, F31, E35, R36, L39, F40, I42, S43, S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78, R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275, V276, R277, F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379.
The avtive spot district
The avtive spot district be have at least one and catalysis triplet (residue H193, D242, S344) in the spacing of any atom exist
Figure C01804864D0058101606QIETU
Any residue of the atom in the scope: I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379, V380, Q382, Y383, W386, L387, L400 and F405.
The ridge of avtive spot engagement groove
The ridge of avtive spot engagement groove is defined as by the visual observations to FVIIa structure 1FAK.pdb: N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370.
Embodiment 2
Be designed for the expression cassette of expressing human proconvertin in mammalian cell
DNA sequence shown in the synthetic SEQ ID NO:2 (comprising the short type (Hagen etc., 1986.PNAS 83:2412) that coding has the human blood coagulation factor VII full-length cDNA of natural short signal peptide) is to promote its high expressed in mammalian cell.At first according to Kozak consensus sequence (Kozak, M.J Mol Biol1987 Aug 20; 196 (4): the 947-50) sequence on every side of modification ATG start codon, so that ATG upstream from start codon consensus sequence mates fully.Then, the open reading frame of natural human thrombin cDNA is modified, making employed codon is the codon of using always in the people's gene of high expressed.Subsequently, at two translation stop codon of the terminal insertion of open reading frame, so that translation effectively stops.The FVII gene of and optimization expression synthetic fully with 70 aggressiveness DNA oligonucleotide assemblings with inserting 5 respectively ' and the terminal primer in 3 ' terminal BamHI and HindIII site, carries out the pcr amplification of standard at last, obtains following sequence (SEQ ID NO:4):
ggatcccgccaccatggtcagccaggccctccgcctcctgtgcctgctcctggggctgcagggctgcctggctgccgtcttcgtcaccca
ggaggaagcccatggcgtcctgcatcgccggcgccgggccaatgcctttctggaagagctccgccctggctccctggaacgcgaatgc
aaagaggaacagtgcagctttgaggaagcccgggagattttcaaagacgctgagcggaccaaactgttttggattagctatagcgatggc
gatcagtgcgcctccagcccttgccagaacgggggctcctgcaaagaccagctgcagagctatatctgcttctgcctgcctgcctttgagg
ggcgcaattgcgaaacccataaggatgaccagctgatttgcgtcaacgaaaacgggggctgcgagcagtactgcagcgatcacacggg
cacgaagcggagctgccgctgccacgaaggctatagcctcctggctgacggggtgtcctgcacgcccacggtggaatacccttgcggg
aagattcccattctagaaaagcggaacgctagcaaaccccagggccggatcgtcggcgggaaggtctgccctaagggggagtgcccct
ggcaggtcctgctcctggtcaacggggcccagctgtgcggcgggaccctcatcaataccatttgggtcgtgtccgccgctcactgcttcg
ataagattaagaattggcggaacctcatcgctgtgctcggcgaacacgatctgtccgagcatgacggggacgaacagtcccgccgggtg
gctcaggtcatcattccctccacctatgtgcctggcacgaccaatcacgatatcgctctgctccgcctccaccagcccgtcgtgctcaccga
tcacgtcgtgcctctgtgcctgcctgagcggacctttagcgaacgcacgctggctttcgtccgctttagcctcgtgtccggctggggccag
ctgctcgaccggggcgctaccgctctcgagctgatggtgctcaacgtcccccggctgatgacccaggactgcctgcagcagtcccgcaa
agtgggggactcccccaatatcacggagtatatgttttgcgctggctatagcgatggctccaaggatagctgcaagggggactccggcg
ggccccatgccacgcactatcgcgggacctggtacctcaccgggatcgtcagctggggccagggctgcgccacggtggggcactttg
gcgtctacacgcgcgtcagccagtacattgagtggctgcagaagctcatgcggagcgaaccccggcccggggtgctcctgcgggccc
ctttcccttgataaaagctt
By preparing the cloning vehicle that carries out that is used for gained PCR product from pCINeo (Promega) clone intron, this PCR product comprises the expression cassette of natural human proconvertin.The synthetic intron of pCI-Neo increases with above-mentioned Standard PC R condition, and primer is as follows:
CBProFpr174：5′-AGCIGGCTAGCCACIGGGCAGGIAAGIAICA-3′
CBProFpr175：5′-TGGCGGGATCCTTAAGAGCTGTAATTGAACT-3′
Produce a 322bp PCR fragment.This fragment is sheared with NheI and BamHI, is cloned into pCDNA3.1/HygR (from Invitrogen) afterwards, produces PF#34.
Clone PF#34 goes up the expression cassette of the natural human proconvertin between BamHI and the HindIII site, obtains plasmid PF#226.
Embodiment 3
Make up the expression cassette of the variant form of coding human blood coagulation factor VII, this variant has glycosylation site in the other body
Extend the construct that (SOE) PCR produces the human blood coagulation variant open reading frame with the codon that has replaced with the sequence jag.In SOE-PCR, the N-end portion of FVII open reading frame and C-end portion are all at first obtaining amplification among the elementary PCR separately.
For example, for R315 and V317 codon being changed into N315 and T317 codon, pairing primer below in elementary PCR, using:
CBProFpr216：5′-CTTAAGGATCCCGCCACCATGGTCAGCCAG-3′
CBProFpr229:5 '-GGAGTCCCCGGTTTTGTTGGACTGCTGC-3 ', and
CBProFpr221：5′-ACTT AAGCTTTTATCAAGGGA-3′
CBProFpr228：5′-GCAGCAGTCCAACAAAACCGGGGACTCC-3′
Make up elementary PCR product then, add terminal primer (CBProFpr216 and CBProFpr221), produce the secondary full length product of the R315N+V317T FVII variant of coding expectation.This secondary PCR product is sheared with BamHI and HindIII, is cloned between the BamHI and HindIII site of carrier PF#34 again, produces PF#249.
In addition, when the unique restriction endonuclease sites close enough in sudden change of introducing and the expression plasmid, can make up variant gene, described construction method comprises a single PCR step and follow-up clone.For example, in a single PCR reaction, use following PCR primer to introduce and replace K143N+N145T:
CBProFpr226：5′-CATTCTAGAAAACCGGACCGCTAGCAAACC-3′
CBProFpr221：5′-ACTTAAGCTTTTATCAAGGGA-3′
Then by using restriction endonuclease sites XbaI and HindIII clone clone gained PCR product.
Use above strategy, can prepare following glycosylation conjugate, the detection of its amide decomposition activity is as detecting described in the low-level catalytic activity chapters and sections.In addition, some conjugates are detected the described step coagulation analysis of blood coagulation activity chapters and sections.Gained as a result editor in following table.
The glycosylation conjugate Amide decomposition activity Blood coagulation activity
T106N K143N+N145T V253N R290N+A292T G291N R315N+V317T K143N+N145T+R315N+V317T +++- - ++ + ndndndnd+ nd
+: detectable activity;-: undetectable activity; Nd: undetermined
Embodiment 4
Introduce other nearside (being positioned at the N-end) glycosylation site to improve the purposes of glycosylation site
In order to prevent self cracking of wild type people FVII, replace by producing R315N and V317T as mentioned above, 315 places introduce a glycosylation site in the position, obtain PF#249.
With Lipofactamine 2000 transfection CHO K1 cells, obtain low-level transient expression.Utilize the amide decomposition run,
Figure C01804864D00611
FVII analyzes 24 hours instantaneous supernatant, shows that this variant is activated.After selecting, obtain the stable clone group with 400 μ g/ml HYGs.These clones express the R315N+V317T variant with the concentration of about 0.2 μ g/ml, can carry out the protein blot analysis under this concentration, with the degree of utilizing of definite glycosylation site of introducing.With the 24 hour supernatants of western blot analysis from the stable clone group, the result is presented at the glycosylation site of introducing at 315 places, position and is partly utilized, and makes an appointment with secretory protein after half the processing fully by glycosylation.Yet if 143 places are transferred to by producing the K143N+N145T replacement in the Natively glycosylated site at 145 places, protein blot shows that the glycosylation site of 315 introducings is by glycosylation fully.
Embodiment 5
In the HEK293 cell, express FVII
That cultivates inoculation 20% in the T-25 culture bottle converges cell line HEK293 (ATCC#CRL-1573), and used culture medium is DMEM, high glucose 10% heat-inactivated FCS (Gibco/BRL Cat#10091), and 5 μ g/ml plant quinone, make the cell growth up to converging.The cell monolayer that converges is with 1,2, and the above-mentioned plasmid p226 of 5,10 and 20 μ g is that transfection reagent (Life technologies) carries out transfection according to manufacturer's method with Lipofectamine 2000.Culture fluid taken a sample in 24 hours after the transfection.The concentration average out to 0.15 μ g/ml of FVII in 24 hours transient expression experiments.
Subsequently, give the selection culture medium that cell comprises 100 μ g/ml HYGs.Upgraded a subculture in per 3 or 4 days, after 3 weeks were selected, in the culture bottle with 1 μ g plasmid DNA and 2 μ g plasmid DNA transfections, the hygromycin resistance cell had grown to and has converged.Collect the cell of each bottle in five culture bottles and gather.Obtain to express the stable transfection subgroup of natural human proconvertin, it is freezing in liquid nitrogen according to standard method.
Embodiment 6
The HEK293 cell of preparation stably express FVII
One bottle HEK293PF#226 transfectant is thawed, and with cell inoculation in containing the high glucose of 15ml DMEM, 10%FCS, plant quinone (5 μ g/ml), 100 U/l penicillins, 75 cm2 tissue culture flasks of 100 μ g/l streptomycins (it is used for all experiments afterwards) were grown 24 hours.Collecting cell is spread 96 hole microtitre flat boards with its dilution and with the density of 1/2 cells/well.After 12 days, the colony of about 20-100 cell comes across in the hole, and labelling is carried out in those holes that only comprise a colony.After the regrowth 2 days, in institute is porose, add 100 μ l culture medium.Two days later, change culture medium in all holes that only contain a colony.First colony was transferred to 25cm after 3 days 2Cultivate in the tissue culture flasks,, colony is transferred to 25cm at ensuing 11 days according to converging degree 2Cultivate in the tissue culture flasks.When in the T-25 tissue culture flasks, growing into when converging, change culture medium, make the clone continue FVII to be secreted in the growth medium in 24 hours, collect supernatant then, detect the existence of factor VII with COASET FVII amide decomposition run.Discovery has a clone, and C18 expresses the FVII of 29 μ g/ml.
Embodiment 7
The expression of FVII glycosylation variant, described variant does not have amide decomposition activity, can suppress the function of FVIIa
The expression plasmid that is used for expression activity site ridge mutant R290N+A292T and G291N, the method described in the embodiment 3 of can pressing substantially makes up.
Use the amide decomposition run
Figure C01804864D00621
FVII (referring to above-mentioned) assesses two kinds of FVII glycosylation variants (R290N+A292T and G291N) and suppresses the active ability of rFVIIa.With Lipofectamin 2000 will encode the plasmid PF#250 of R290N+A292T and coding G291N plasmid PF#294 transfection near converge in the HEK293 cell that serum is cultivated.After the transfection, with the CO of transfectional cell at 37 ℃ 5% 2Following incubation 3 hours, (Ex-Cyte plants quinone, P/S) for DMEM, ITS-A then culture medium to be replaced by serum-free medium.After 40 hours of transfection, collect culture fluid and centrifugally make its clarification, in order to analyzing.
Use rFVIIa:0.0125ng, 0.025ng, 0.05ng, 0.075ng and 0.1ng, production standard curve.Add 0.025ng rFVIIa in the culture fluid of any the 50 μ l among two kinds of non-activity glycosylation variant R290N+A292T and the G291N are undiluted, 2 times of dilutions, 5 times of dilutions, 10 times of dilutions and 50 times of dilutions or the culture fluid of simulation transfection.In COASET FVII test, the FVIIa of 0.025ng is equivalent to OD 405=0.35 (first round) and OD 405The signal of=0.26 (second takes turns).
With the editor as a result that obtains in following table:
Figure C01804864D00631
Above-mentioned data show glycosylation conjugate G291N and R290N+A292T suppress the function of rFVIIa.
Embodiment 8
The purification of FVII and activation afterwards
The purification of wild type factor VII and its conjugate carries out at 4 ℃.The supernatant that obtains from the cell of expressing described conjugate (or wild type FVII) dilutes twice through aseptic filtration (0.22 μ m) in cold ultrapure (milli Q) water.Add EDTA to 5mM, pH is adjusted to 8.6, conductivity is lower than 10mS/cm.Sample is loaded in 4 ℃ of quick stir-in resins of the equilibrated Q-Sepharose of the Tris with 10mM pH8.6 (Phamacia).Behind last sample, with 10mM Tris (pH8.6), 150mM NaCl washs chromatographic column, reaches baseline value up to the light absorption of 280nm.Then at 10mM Tris (pH8.6), this chromatographic column of balance among the 100mMNaCl.Conjunction type conjugate (or wild type FVII) 10mM Tris (pH8.6), 100mMNaCl, 5mM CaCl 2Eluting.Compile the fraction that is rich in conjugate (or wild type FVII), dialysis or use Vivaspin enrichment facility (Vivascience) concentrate.
Oneself's activation of conjugate (or wild type FVII) is passed through at 10mM Tris (pH7.8-8.6), 100mM NaCl, 5mM CaCl 2In concentrate and the albumen of incubation institute eluting obtains.
In addition, under 37 ℃, 10mM Tris (pH7.4-8.0), 100mM NaCl, 5mM CaCl 2In, can activate described conjugate (or wild type FVII) with the link coupled factor Xa of the activatory Sepharose of CNBr-.
Conjugate (or wild type FVIIa) transferred to by buffer-exchanged contain 10mM CaCl 2, 50mM NaCl, 3% mannitol, 0.05%Tween80, in the pH5.6 solutions buffered, filtration sterilization is ℃ storage also-80.
Embodiment 9
The terminal PEGization of the N-of FVII
PH5.5 contain the 10mM sodium citrate, 20mM calcium chloride is in the pH5.5 buffer of 100mm sodium chloride, with methoxy poly (ethylene glycol) (mPEG) coupling of M-PEG-CHO (M-ALD-5000 is from Shearwater) with factor FVII and the about 5kDa of molecular weight.M-PEG-CHO exists with excessive 50-100 molar concentration doubly, and described protein concentration is 0.2-0.5mg/ml.Reaction is carried out with 300-1500 μ l amount in batches, at room temperature stirs 1 hour at every turn, adds excessive 500-1000 NaBH3CN doubly, continues the incubation that spends the night, and room temperature is stirred.
The FVII of PEGization through buffer-exchanged transfer to buffer A (10mM Tris, pH7.6), and 4 ℃ with it on sample to the mono Q post (Pharmacia) that balance has been crossed in buffer A.With from the gradient of 0-100%B (10mM Tris, (pH7.6), 500mM NaCl) with 40 times of column volume elution of bound type albumen.
Embodiment 10
Rat Chinese medicine dynamics research
All (pH5.5 contains 1.5mg/ml CaCl at 1.3mg/ml glycyl-glycine buffer for wild type FVII and conjugate of the present invention 2, 30mg/ml mannitol, 0.1mg/ml polysorbat 80 and 3mg/ml NaCl) and middle preparation.In order to determine half life in the body, each preparation gives S-D (Sprague-Dawley) rat with quick (bolus) injection in the disposable vein.Be injected in about 10 seconds and slowly give, to reduce the potential heart failure danger that causes because of high Ca++ concentration.From the rat that 9 anesthesia are put to death,, as injected back 1 minute with proper spacing, 15 minutes, 30 minutes, 45 minutes and 1 hour, blood sample collection.With described blood sample collection in the 1ml test tube that contains adenine (Sigma#C4431) and 50 μ l citric acid-phosphoric acid-glucose solutions to avoid blood coagulation.Immediately sample is stored in after the sampling about 0 ℃ up to centrifugal, collect Citrated blood plasma supernatant then and be used for analyzing.Sample is analyzed by a step thrombotest described in the method chapters and sections that detect blood coagulation activity, 1 calculates half life then.

Claims (21)

1.一种多肽偶联物，其包含：1. A polypeptide conjugate comprising: (a)多肽，所述多肽包含这样的氨基酸序列，所述氨基酸序列与SEQ IDNO：1所示野生型人FVII或FVIIa的氨基酸序列有1-15个氨基酸残基不同并且相对于SEQ ID NO：1包含至少一个引入的体内N-糖基化位点，所述引入的体内N糖基化位点具有序列N-X-S/T/C，其中X是除脯氨酸之外的任何氨基酸残基，N是天冬酰胺，S/T/C是丝氨酸、苏氨酸或半胱氨酸，和(a) a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of wild-type human FVII or FVIIa shown in SEQ ID NO: 1 by 1-15 amino acid residues and relative to SEQ ID NO: 1 comprising at least one introduced in vivo N-glycosylation site having the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine, S/T/C is serine, threonine, or cysteine, and (b)至少一个与引入的体内N-糖基化位点共价连接的糖组分，(b) at least one sugar component covalently linked to the introduced in vivo N-glycosylation site, 其中所述偶联物显示人FVIIa的至少25％的凝血活性。wherein said conjugate exhibits at least 25% of the coagulation activity of human FVIIa. 2.权利要求1的偶联物，其中所述体内N-糖基化位点通过取代而引入。2. The conjugate of claim 1, wherein the in vivo N-glycosylation site is introduced by substitution. 3.权利要求1或2的偶联物，其中所述引入的体内N-糖基化位点被引入由有25％以上的侧链暴露于溶剂的氨基酸残基所占据的位置。3. The conjugate according to claim 1 or 2, wherein the introduced in vivo N-glycosylation site is introduced at a position occupied by an amino acid residue with more than 25% of the side chain exposed to the solvent. 4.权利要求3的偶联物，其中所述引入的体内N-糖基化位点被引入由有50％以上的侧链暴露于溶剂的氨基酸残基所占据的位置。4. The conjugate of claim 3, wherein the introduced in vivo N-glycosylation site is introduced at a position occupied by an amino acid residue with more than 50% of its side chain exposed to solvent. 5.权利要求1-4中任一项的偶联物，包含至少一种选自下组的取代：5. The conjugate of any one of claims 1-4, comprising at least one substitution selected from the group consisting of: F4S/T，P10N，Q21N，W41N，S43N，A51N，G58N，L65N，G59S/T，E82S/T，N95S/T，G97S/T，Y101N，D104N，T106N，K109N，G117N，G124N，S126N，T128N，I186S/T，R202S/T，I205S/T，D212N，E220N，V253N，E265N，T267N，E270N，R277N，L280N，P303S/T，L305N，Q312N，G318N，G331N，D334N，K337N，R353N，Y357N，和M391N。F4S/T, P10N, Q21N, W41N, S43N, A51N, G58N, L65N, G59S/T, E82S/T, N95S/T, G97S/T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N, T128N, I186S/T, R202S/T, I205S/T, D212N, E220N, V253N, E265N, T267N, E270N, R277N, L280N, P303S/T, L305N, Q312N, G318N, G331N, D334N, K337N, R353N, and M353N, Y359N . 6.权利要求5的偶联物，包含至少一种选自下组的取代：6. The conjugate of claim 5, comprising at least one substitution selected from the group consisting of: F4S/T，P10N，Q21N，W41N，A51N，G58N，G59S/T，N95S/T，G97S/T，Y101N，D104N，T106N，K109N，G117N，G124N，S126N，T128N，I186S/T，R202S/T，I205S/T，D212N，E220N，V253N，E265N，T267N，E270N，L280N，P303S/T，G318N，G331N，D334N，K337N，R353N，Y357N和M391N。F4S/T, P10N, Q21N, W41N, A51N, G58N, G59S/T, N95S/T, G97S/T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N, T128N, I186S/T, R202S/T, I205S/T, D212N, E220N, V253N, E265N, T267N, E270N, L280N, P303S/T, G318N, G331N, D334N, K337N, R353N, Y357N and M391N. 7.权利要求6的偶联物，包含至少一种选自由T106N，I205S/T和V253N组成的组中的取代。7. The conjugate of claim 6, comprising at least one substitution selected from the group consisting of T106N, I205S/T and V253N. 8.权利要求7的偶联物，包含至少一种选自由T106N和V253N组成的组中的取代。8. The conjugate of claim 7, comprising at least one substitution selected from the group consisting of T106N and V253N. 9.权利要求1-4之一的偶联物，其中所述体内N-糖基化位点的引入位置是，不形成活性位点区或活性位点结合槽的脊的一部分的位置，所述活性位点区定义为残基I153，Q167，V168，L169，L170，L171，Q176，L177，C178，G179，G180，T181，V188，V189，S190，A191，A192，H193，C194，F195，D196，K197，I198，W201，V228，I229，I230，P231，S232，T233，Y234，V235，P236，G237，T238，T239，N240，H241，D242，I243，A244，L245，L246，V281，S282，G283，W284，G285，Q286，T293，T324，E325，Y326，M327，F328，D338，S339，C340，K341，G342，D343，S344，G345，G346，P347，H348，L358，T359，G360，I361，V362，S363，W364，G365，C368，V376，Y377，T378，R379，V380，Q382，Y383，W386，L387，L400和F405，所述活性位点结合槽的脊定义为N173，A175，K199，N200，N203，D289，R290，G291，A292，P321和T370。9. The conjugate of any one of claims 1-4, wherein the introduction position of the N-glycosylation site in the body is a position that does not form a part of the ridge of the active site region or the active site binding groove, so The active site region is defined as residues I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196 , K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283 , W284, G285, Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362 , S363, W364, G365, C368, V376, Y377, T378, R379, V380, Q382, Y383, W386, L387, L400 and F405, the ridge of the active site binding groove is defined as N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370. 10.权利要求9的偶联物，其中所述体内N-糖基化位点被引入不形成组织因子结合位点的一部分的位置，所述组织因子结合位点定义为残基L13，K18，F31，E35，R36，L39，F40，I42，S43，S60，K62，D63，Q64，L65，I69，C70，F71，C72，L73，P74，F76，E77，G78，R79，E82，K85，Q88，I90，V92，N93，E94，R271，A274，F275，V276，R277，F278，R304，L305，M306，T307，Q308，D309，Q312，Q313，E325和R379。10. The conjugate of claim 9, wherein said in vivo N-glycosylation site is introduced at a position that does not form part of the tissue factor binding site defined by residues L13, K18, F31, E35, R36, L39, F40, I42, S43, S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78, R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275, V276, R277, F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379. 11.权利要求9或10的偶联物，其中所述取代选自下组：11. The conjugate of claim 9 or 10, wherein the substitution is selected from the group consisting of: K32N+A34S/T，I30N+K32S/T，W41N，Y44N+D46S/T，S45N+G47S/T，D46N+D48S/T，G47N+Q49S/T，K143N+N145S/T，E142N+R144S/T，L141N+K143S/T，I140N+E142S/T，R144N+A146S/T，A146N+K148S/T，S147N+P149S/T，R315N+V317S/T，S314N+K316S/T，K316N+G318S/T，V317N+D319S/T，G318N，R392N+E394S/T，M391N，L390N+R392S/T，K389N+M391S/T，S393N+P395S/T，E394N+R396S/T，P395N+P397S/T，R396N+G398S/T，P397N+V399S/T，V399N+L401S/T，L401N+A403S/T，R402N+P404S/T，P404N+P406S/T和K143N+N145S/T+R315N+V317S/T。K32N+A34S/T, I30N+K32S/T, W41N, Y44N+D46S/T, S45N+G47S/T, D46N+D48S/T, G47N+Q49S/T, K143N+N145S/T, E142N+R144S/T, L141N+K143S/T, I140N+E142S/T, R144N+A146S/T, A146N+K148S/T, S147N+P149S/T, R315N+V317S/T, S314N+K316S/T, K316N+G318S/T, V317N+ D319S/T, G318N, R392N+E394S/T, M391N, L390N+R392S/T, K389N+M391S/T, S393N+P395S/T, E394N+R396S/T, P395N+P397S/T, R396N+G398S/T, P397N+V399S/T, V399N+L401S/T, L401N+A403S/T, R402N+P404S/T, P404N+P406S/T and K143N+N145S/T+R315N+V317S/T. 12.权利要求11的偶联物，其中所述取代选自下组：K32N+A34T，Y44N+D46T，K143N+N145T，R315N+V317T，R392N+E394T，R396N+G398T，R402N+P404T，和K143N+N145T+R315N+V317T。12. The conjugate of claim 11, wherein the substitution is selected from the group consisting of K32N+A34T, Y44N+D46T, K143N+N145T, R315N+V317T, R392N+E394T, R396N+G398T, R402N+P404T, and K143N+ N145T+R315N+V317T. 13.多肽，其具有权利要求1-12中任一项中定义的多肽。13. Polypeptide having a polypeptide as defined in any one of claims 1-12. 14.一种核苷酸序列，其编码权利要求13的多肽。14. A nucleotide sequence encoding the polypeptide of claim 13. 15.一种表达载体，其包含权利要求14的核苷酸序列。15. An expression vector comprising the nucleotide sequence of claim 14. 16.一种宿主细胞，其包含权利要求14的核苷酸序列或权利要求15的表达载体。16. A host cell comprising the nucleotide sequence of claim 14 or the expression vector of claim 15. 17.权利要求16的宿主细胞，其中所述宿主细胞是可进行体内糖基化的γ-羧基化细胞。17. The host cell of claim 16, wherein said host cell is a gamma-carboxylation cell capable of glycosylation in vivo. 18.一种药物组合物，其包括权利要求1-12中任一项所定义的偶联物和一种可药用的载体或赋形剂。18. A pharmaceutical composition comprising the conjugate as defined in any one of claims 1-12 and a pharmaceutically acceptable carrier or excipient. 19.权利要求1-12中任一项所定义的偶联物，所述偶联物用做药物。19. A conjugate as defined in any one of claims 1-12 for use as a medicament. 20.权利要求1-12中任一项所定义的偶联物的用途，用于生产治疗哺乳动物FVIIa/TF相关疾病的药物。20. Use of the conjugate as defined in any one of claims 1-12 for the manufacture of a medicament for the treatment of FVIIa/TF-related diseases in mammals. 21.权利要求18的用途，其中所述FVIIa/TF相关疾病选自需要加强凝血的疾病。21. The use of claim 18, wherein the FVIIa/TF-associated disease is selected from diseases requiring enhanced coagulation.
CNB018048641A 2000-02-11 2001-02-12 Coagulation factor VII or VIIa-like molecules Expired - Fee Related CN100488562C (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA200000218 2000-02-11
DKPA200000218 2000-02-11
DKPA200001558 2000-10-18
DKPA200001558 2000-10-18

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CNA2009101282432A Division CN101503726A (en) 2000-02-11 2001-02-12 Factor VII or VIIa-like molecules

Publications (2)

Publication Number Publication Date
CN1400910A CN1400910A (en) 2003-03-05
CN100488562C true CN100488562C (en) 2009-05-20

Family

ID=26068765

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB018048641A Expired - Fee Related CN100488562C (en) 2000-02-11 2001-02-12 Coagulation factor VII or VIIa-like molecules

Country Status (18)

Country Link
US (13) US6806063B2 (en)
EP (3) EP2319541A1 (en)
JP (2) JP2003521930A (en)
CN (1) CN100488562C (en)
AT (1) ATE428445T1 (en)
AU (3) AU783512B2 (en)
CA (2) CA2397347C (en)
CY (1) CY1109614T1 (en)
DE (1) DE60138364D1 (en)
DK (1) DK1257295T3 (en)
ES (1) ES2325877T3 (en)
IL (2) IL150692A0 (en)
NO (1) NO20023804L (en)
NZ (1) NZ521257A (en)
PL (2) PL204285B1 (en)
PT (1) PT1257295E (en)
RU (1) RU2278123C2 (en)
WO (1) WO2001058935A2 (en)

Families Citing this family (168)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7247708B2 (en) * 1997-10-23 2007-07-24 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US6747003B1 (en) 1997-10-23 2004-06-08 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
WO2001004287A1 (en) * 1999-07-07 2001-01-18 Maxygen Aps A method for preparing modified polypeptides
CN100488562C (en) * 2000-02-11 2009-05-20 拜耳医药保健有限公司 Coagulation factor VII or VIIa-like molecules
US7220837B1 (en) 2000-04-28 2007-05-22 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US7812132B2 (en) 2000-04-28 2010-10-12 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US6905683B2 (en) 2000-05-03 2005-06-14 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
US6423826B1 (en) * 2000-06-30 2002-07-23 Regents Of The University Of Minnesota High molecular weight derivatives of vitamin K-dependent polypeptides
US20030211094A1 (en) * 2001-06-26 2003-11-13 Nelsestuen Gary L. High molecular weight derivatives of vitamin k-dependent polypeptides
US7160540B2 (en) 2000-06-30 2007-01-09 Regents Of The University Of Minnesota Methods for detecting activity of clottings factors
US7176288B2 (en) * 2000-09-13 2007-02-13 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
JP4361728B2 (en) * 2000-09-13 2009-11-11 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Human coagulation factor VII mutant
AU2002218029A1 (en) * 2000-11-09 2002-05-21 The Scripps Research Institute Modified factor viia
US7235638B2 (en) 2001-03-22 2007-06-26 Novo Nordisk Healthcare A/G Coagulation factor VII derivatives
JP2004529640A (en) * 2001-03-22 2004-09-30 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Coagulation factor VII derivative
AU2002333211B2 (en) * 2001-09-27 2008-05-08 Novo Nordisk Health Care Ag Human coagulation factor VII polypeptides
US7052868B2 (en) 2001-09-27 2006-05-30 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
CN105131104B (en) * 2001-10-10 2018-11-16 诺和诺德公司 The reconstruct and sugar conjugation of peptide
US7157277B2 (en) 2001-11-28 2007-01-02 Neose Technologies, Inc. Factor VIII remodeling and glycoconjugation of Factor VIII
US7795210B2 (en) 2001-10-10 2010-09-14 Novo Nordisk A/S Protein remodeling methods and proteins/peptides produced by the methods
US7696163B2 (en) 2001-10-10 2010-04-13 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US7173003B2 (en) 2001-10-10 2007-02-06 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
US7214660B2 (en) 2001-10-10 2007-05-08 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
JP2005512524A (en) 2001-11-02 2005-05-12 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Human coagulation factor VII polypeptide
US6960657B2 (en) 2001-11-02 2005-11-01 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7125846B2 (en) 2001-11-09 2006-10-24 Novo Nordisk Healthcare A/G Pharmaceutical composition comprising factor VII polypeptides and factor V polypeptides
KR20040065278A (en) * 2001-12-21 2004-07-21 노보 노르디스크 에이/에스 Liquid composition of modified factor vii polypeptides
ES2325653T3 (en) 2001-12-21 2009-09-11 Novo Nordisk Health Care Ag LIQUID COMPOSITION OF FACTOR POLYPEPTIDES VII.
CA2478522A1 (en) * 2002-03-08 2003-09-18 Emory University Novel curcuminoid-factor viia constructs as suppressors of tumor growth and angiogenesis
US20050069551A1 (en) * 2002-03-08 2005-03-31 Emory University Cytotoxic compound-protein conjugates as suppressors of tumor growth and angiogenesis
MXPA04010781A (en) * 2002-04-30 2005-03-07 Maxygen Holdings Ltd FACTOR VII OR VIIa POLYPEPTIDE VARIANTS.
US20040009918A1 (en) * 2002-05-03 2004-01-15 Hanne Nedergaard Stabilised solid compositions of modified factor VII
DK1517698T4 (en) 2002-06-21 2018-01-29 Novo Nordisk Healthcare Ag Stabilized solid compositions of Factor VIIa polypeptides
WO2004000366A1 (en) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Pegylated factor vii glycoforms
MXPA04012496A (en) 2002-06-21 2005-09-12 Novo Nordisk Healthcare Ag Pegylated factor vii glycoforms.
DE60330923D1 (en) * 2002-09-25 2010-02-25 Novo Nordisk Healthcare Ag Human coagulation factor VII polypeptides
US6911323B2 (en) 2002-09-25 2005-06-28 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
CA2502162C (en) * 2002-09-30 2014-04-15 Bayer Healthcare Llc Fvii or fviia variants having increased clotting activity
AU2004221824B2 (en) 2003-03-14 2009-08-27 Ratiopharm Gmbh Branched water-soluble polymers and their conjugates
WO2004082708A2 (en) * 2003-03-18 2004-09-30 Novo Nordisk Health Care Ag Liquid, aqueous, pharmaceutical compositions of factor vii polypeptides
BRPI0408417A (en) * 2003-03-18 2006-03-21 Novo Nordisk Healthcare Ag methods for producing a serine protease-containing gla residue, for purifying a serine protease-containing gla residue and for stabilizing a serine protease-containing gla residue;
ES2327044T3 (en) * 2003-03-20 2009-10-23 Bayer Healthcare Llc VARIATIONS OF FVII OR FVIIA.
US7897734B2 (en) 2003-03-26 2011-03-01 Novo Nordisk Healthcare Ag Method for the production of proteins
SG155777A1 (en) 2003-04-09 2009-10-29 Neose Technologies Inc Glycopegylation methods and proteins/peptides produced by the methods
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
WO2004091499A2 (en) 2003-04-09 2004-10-28 Neose Technologies, Inc. Intracellular formation of peptide conjugates
US7348004B2 (en) 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
EP3002012A1 (en) 2003-05-06 2016-04-06 Biogen Hemophilia Inc. Clotting factor-fc chimeric proteins to treat hemophilia
TWI353991B (en) 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
JP2007523630A (en) 2003-05-09 2007-08-23 ネオス テクノロジーズ インコーポレイテッド Composition and formulation of human growth hormone glycosylation mutants
KR20060015574A (en) * 2003-05-23 2006-02-17 노보 노르디스크 헬스 케어 악티엔게젤샤프트 Protein Stabilization in Solution
MXPA05013769A (en) * 2003-06-19 2006-03-08 Maxygen Holdings Ltd FACTOR VII OR VIIa GLA DOMAIN VARIANTS.
WO2004112828A1 (en) 2003-06-25 2004-12-29 Novo Nordisk Health Care Ag Liquid composition of factor vii polypeptides
JP5306597B2 (en) * 2003-07-01 2013-10-02 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Liquid aqueous pharmaceutical composition of factor VII polypeptide
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
WO2005025623A2 (en) * 2003-07-28 2005-03-24 Emory University Ef-24-factor vii conjugates
WO2005014049A2 (en) * 2003-08-08 2005-02-17 Novo Nordisk A/S Synthesis and application of new structural well defined branched polymers as conjugating agents for peptides
US20060198819A1 (en) * 2003-08-08 2006-09-07 Novo Nordisk Healthcare A/G Use of galactose oxidase for selective chemical conjugation of protractor molecules to proteins of therapeutic interest
RU2388460C2 (en) * 2003-08-14 2010-05-10 Ново Нордиск Хелс Кеа Аг Liquid water pharmaceutical composition containing factor vii polypeptide
EP1664291B1 (en) * 2003-09-09 2012-02-29 Novo Nordisk Health Care AG Coagulation factor vii polypeptides
CN101870729A (en) * 2003-09-09 2010-10-27 诺和诺德医疗保健公司 Coagulation factor vii polypeptides
EP1673453A2 (en) 2003-10-07 2006-06-28 Novo Nordisk Health Care AG Hybrid molecules having factor vii/viia activity
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
ES2507091T3 (en) 2003-12-01 2014-10-14 Novo Nordisk Health Care Ag Nanofiltration of factor VII solutions to eliminate viruses
US20060040856A1 (en) 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
KR20140093711A (en) 2003-12-19 2014-07-28 노보 노르디스크 헬스 케어 악티엔게젤샤프트 Stabilised compositions of factor vii polypeptides
ES2560657T3 (en) 2004-01-08 2016-02-22 Ratiopharm Gmbh O-linked glycosylation of G-CSF peptides
CA2552043A1 (en) * 2004-01-21 2005-08-04 Novo Nordisk A/S Transglutaminase mediated conjugation of peptides
KR100545720B1 (en) * 2004-05-31 2006-01-24 메덱스젠 주식회사 Glycated immunoglobulins and immunoconjugates comprising them
WO2005123916A2 (en) * 2004-06-21 2005-12-29 Novo Nordisk Health Care Ag Glycosylation-disrupted factor vii variants
WO2006010143A2 (en) 2004-07-13 2006-01-26 Neose Technologies, Inc. Branched peg remodeling and glycosylation of glucagon-like peptide-1 [glp-1]
US20090176967A1 (en) * 2004-08-02 2009-07-09 Novo Nordisk Healthcare A/G Conjugation of FVII
ATE469216T1 (en) * 2004-08-17 2010-06-15 Csl Behring Gmbh MODIFIED VITAMIN K DEPENDENT POLYPEPTIDES
EP1799249A2 (en) 2004-09-10 2007-06-27 Neose Technologies, Inc. Glycopegylated interferon alpha
PL2586456T3 (en) 2004-10-29 2016-07-29 Ratiopharm Gmbh Remodeling and glycopegylation of fibroblast growth factor (FGF)
MX350293B (en) * 2004-11-12 2017-09-04 Bayer Healthcare Llc Site-directed modification of fviii.
AU2005318106B2 (en) 2004-12-23 2011-11-03 Novo Nordisk Health Care Ag Reduction of the content of protein contaminants in compositions comprising a Vitamin K-dependent protein of interest
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
EP1871795A4 (en) 2005-04-08 2010-03-31 Biogenerix Ag Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
EP1893230A2 (en) * 2005-04-26 2008-03-05 Maxygen Holdings Ltd. Use of modified factor vii for treating bleeding
EP1891231A4 (en) 2005-05-25 2011-06-22 Novo Nordisk As FACTOR IX GLYCOPEGYL
WO2006127910A2 (en) 2005-05-25 2006-11-30 Neose Technologies, Inc. Glycopegylated erythropoietin formulations
WO2006134173A2 (en) 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
EP1898945B1 (en) * 2005-06-24 2012-12-19 Drugrecure ApS Airway administration of site-inactivated fviia in inflammatory conditions affecting the respiratory tract
WO2007006808A1 (en) 2005-07-13 2007-01-18 Novo Nordisk Health Care Ag Host cell protein knock-out cells for production of therapeutic proteins
US20070105755A1 (en) 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
US20090017007A1 (en) * 2005-08-26 2009-01-15 Maxygen Holdings Ltd. Liquid factor vii composition
WO2007026021A1 (en) 2005-09-01 2007-03-08 Novo Nordisk Health Care Ag Hydrophobic interaction chromatography purification of factor vii polypeptides
EP2316930A1 (en) 2005-09-14 2011-05-04 Novo Nordisk Health Care AG Human coagulation factor VII polypeptides
WO2007039475A1 (en) * 2005-09-21 2007-04-12 Novo Nordisk Health Care Ag Human coagulation factor vii polypeptides
WO2007056191A2 (en) 2005-11-03 2007-05-18 Neose Technologies, Inc. Nucleotide sugar purification using membranes
EP1816201A1 (en) * 2006-02-06 2007-08-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
ES2399138T3 (en) * 2006-03-16 2013-03-26 Stellaris Pharmaceuticals Aps Local treatment with factor VII
US7985839B2 (en) * 2006-03-31 2011-07-26 Baxter International Inc. Factor VIII polymer conjugates
NZ572050A (en) 2006-03-31 2011-09-30 Baxter Int Factor VIII conjugated to polyethylene glycol
US7645860B2 (en) * 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
US7982010B2 (en) * 2006-03-31 2011-07-19 Baxter International Inc. Factor VIII polymer conjugates
US20110040073A1 (en) * 2006-04-07 2011-02-17 Novo Nordisk Healthcare A/G Covalent Factor VII-Tissue Factor Complex
ES2349024T3 (en) * 2006-04-11 2010-12-21 Csl Behring Gmbh METHOD TO INCREASE THE IN VIVO RECOVERY OF THERAPEUTIC POLYPEPTIDES.
BRPI0712008A2 (en) * 2006-05-24 2012-01-10 Novo Nordisk Healthcare Ag extended fix derivatives and analogs
FR2901707B1 (en) 2006-05-31 2017-09-29 Lab Francais Du Fractionnement RECOMBINANT OR TRANSGENIC FACTOR VII COMPOSITION, EACH FACTOR VII MOLECULE HAVING TWO N-GLYCOSYLATION SITES WITH DEFINED GLYCANNIC PATTERNS
US9175061B2 (en) 2006-07-07 2015-11-03 Novo Nordisk Health Care Ag Protein conjugates and methods for their preparation
WO2008009635A2 (en) * 2006-07-17 2008-01-24 Novo Nordisk Health Care Ag Factor viia analogues with increased activity for treating thrombocytopenia
US20080248959A1 (en) 2006-07-21 2008-10-09 Neose Technologies, Inc. Glycosylation of peptides via o-linked glycosylation sequences
FR2904558B1 (en) * 2006-08-01 2008-10-17 Lab Francais Du Fractionnement "RECOMBINANT OR TRANSGENIC FACTOR VII COMPOSITION, MAJORITYALLY HAVING BIANTENNAE, BISIALYLATED AND NON-FUCOSYLATED GLYCANNIC FORMS"
EP2059527B1 (en) 2006-09-01 2014-12-03 Novo Nordisk Health Care AG Modified glycoproteins
JP2010505874A (en) * 2006-10-03 2010-02-25 ノヴォ ノルディスク アー／エス Purification method for polypeptide conjugates
US8777971B2 (en) * 2006-10-17 2014-07-15 Amj Bv Device and method for joining vessels in anastomosis
CN103804489A (en) 2006-12-15 2014-05-21 巴克斯特国际公司 Factor viia-(poly)sialic acid conjugate having prolonged in vivo half-life
AU2007338298B2 (en) 2006-12-22 2013-02-07 Csl Behring Gmbh Modified coagulation factors with prolonged in vivo half-life
WO2008081024A1 (en) * 2007-01-03 2008-07-10 Novo Nordisk Health Care Ag Subcutaneous administration of coagulation factor viia-related popypeptdes
EP1972687A1 (en) * 2007-03-23 2008-09-24 GenOdyssee Polynucleotides and polypeptides of human factor VII gene, SNPs
BRPI0809670A8 (en) 2007-04-03 2018-12-18 Biogenerix Ag methods to increase stem cell production, to increase the number of granulocytes in an individual, to prevent, treat and alleviate myelosuppression that results from cancer therapy, to treat a condition in an individual, to treat neutropenia and thrombocytopenia in a mammal, to expand hematopoietic stem cells in culture, to increase hematopoiesis in an individual, to increase the number of hematopoietic progenitor cells in an individual, and to provide stable bone marrow graft, and, oral dosage form.
SG174077A1 (en) 2007-04-13 2011-09-29 Catalyst Biosciences Inc Modified factor vii polypetides and uses thereof
US9186323B2 (en) 2007-05-02 2015-11-17 Novo Nordisk Healthcare Ag High concentration factor VII polypeptide formulations comprising an aromatic preservative and an antioxidant
MX2009013259A (en) 2007-06-12 2010-01-25 Novo Nordisk As Improved process for the production of nucleotide sugars.
US20100268055A1 (en) * 2007-07-19 2010-10-21 Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University Self-Anchoring MEMS Intrafascicular Neural Electrode
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
CN102026653B (en) * 2007-10-15 2014-06-18 北卡罗来纳-查佩尔山大学 Human factor IX variant with increased half-life
DK2574677T3 (en) 2007-12-27 2017-11-06 Baxalta GmbH Methods of cell culture
ES2476690T3 (en) 2008-02-27 2014-07-15 Novo Nordisk A/S Factor VIII conjugated molecules
TWI465247B (en) 2008-04-11 2014-12-21 Catalyst Biosciences Inc Modified Factor VII polypeptide and use thereof
CN102105582A (en) 2008-04-21 2011-06-22 诺沃-诺迪斯克有限公司 Hyperglycosylated human coagulation factor IX
BRPI0911350A2 (en) * 2008-04-24 2017-12-05 Celtic Pharma Peg Ltd polyethylene glycol conjugate, method for treating hemophilia b, and method for reducing the risk of disease or conditions.
JP2011520999A (en) * 2008-05-23 2011-07-21 ノボ ノルディスク ヘルス ケア アーゲー Formulation of peg-functionalized serine protease with high concentration of aromatic preservative
CN102083459A (en) * 2008-05-23 2011-06-01 诺沃-诺迪斯克保健股份有限公司 Low viscosity compositions comprising a PEGylated GLA-domain containing protein
KR20110039348A (en) 2008-08-06 2011-04-15 노보 노르디스크 헬스 케어 악티엔게젤샤프트 Conjugated Proteins with Extended In Vivo Efficacy
KR20110122100A (en) 2009-01-22 2011-11-09 노보 노르디스크 헬스 케어 악티엔게젤샤프트 Stable growth hormone compounds
BR122021021381B1 (en) 2009-02-03 2023-05-16 Amunix Pharmaceuticals, Inc METHOD FOR IMPROVING A PROPERTY OF A BIOLOGICALLY ACTIVE PROTEIN
DK2440239T3 (en) 2009-06-09 2017-10-16 Prolong Pharmaceuticals Llc HEMOGLOBIN FORMATIONS
WO2010149172A2 (en) 2009-06-24 2010-12-29 Rigshospitalet SYSTEMIC PRO-HEMOSTATIC EFFECT OF CLOTTING FACTORS IN COMBINATION WITH SYMPATHICOMIMETICS WITH AGONISTIC EFFECTS ON α-ADRENERGIC AND/OR β-ADRENERGIC RECEPTORS OF THE SYMPATHETIC NERVOUS SYSTEM, RELATED TO IMPROVED CLOT STRENGTH.
SG10201401194VA (en) 2009-07-27 2014-07-30 Lipoxen Technologies Ltd Glycopolysialylation of non-blood coagulation proteins
US8809501B2 (en) 2009-07-27 2014-08-19 Baxter International Inc. Nucleophilic catalysts for oxime linkage
KR101912335B1 (en) 2009-07-27 2018-10-26 리폭센 테크놀로지즈 리미티드 Glycopolysialylation of non-blood coagulation proteins
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
KR101759300B1 (en) * 2009-07-27 2017-07-31 리폭센 테크놀로지즈 리미티드 Glycopolysialylation of non-blood coagulation proteins
JP6086528B2 (en) 2009-08-06 2017-03-01 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー Growth hormone with long-term in vivo efficacy
WO2011018515A1 (en) 2009-08-14 2011-02-17 Novo Nordisk Health Care Ag Method of purifying pegylated proteins
JP2013502458A (en) 2009-08-24 2013-01-24 アムニクス オペレーティング インコーポレイテッド Coagulation factor VII composition and methods of making and using the same
US20110150843A1 (en) * 2009-10-30 2011-06-23 National Institute Of Immunology Method for the therapeutic correction of hemophilia a by transplanting bone marrow cells
JP5980689B2 (en) 2010-01-22 2016-08-31 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー Stable growth hormone compound
CN106139158B (en) 2010-01-22 2024-06-21 诺沃—诺迪斯克保健股份有限公司 Growth hormone with prolonged in vivo efficacy
GB201007356D0 (en) 2010-04-30 2010-06-16 Leverton Licence Holdings Ltd Conjugated factor VIIa
KR101331101B1 (en) 2010-06-04 2013-11-19 에스케이케미칼주식회사 Fusion protein having factor vii activity
TWI557135B (en) 2010-11-03 2016-11-11 介控生化科技公司 Modified ninth factor multi-peptide and use thereof
WO2012071631A1 (en) 2010-12-03 2012-06-07 Gene Stream Pty Ltd Improved light-emitting molecules
PL2654794T3 (en) 2010-12-22 2020-07-27 Baxalta GmbH Materials and methods for conjugating a water soluble fatty acid derivative to a protein
HUE043894T2 (en) 2011-06-10 2019-09-30 Bioverativ Therapeutics Inc Pro-coagulant compounds and methods of use thereof
AR088267A1 (en) * 2011-10-06 2014-05-21 Hanmi Science Co Ltd DERIVATIVES, OF THE BLOOD COAGULATION FACTORS VII AND VIIA, CONJUGATES AND COMPLEXES THAT INCLUDE THE SAME AND ITS USES
JP6383666B2 (en) 2012-02-15 2018-08-29 バイオベラティブ セラピューティクス インコーポレイテッド Recombinant factor VIII protein
BR112014020694A2 (en) 2012-02-15 2018-05-08 Amunix Operating Inc. factor viii fusion protein comprising extended recombinant polypeptide (xten) fusion factor polypeptide and its method of manufacture, nucleic acid, vectors, host cell, as well as pharmaceutical composition and its use in the treatment of coagulopathy, bleeding episode and hemophilia a
GB2516388A (en) * 2012-04-16 2015-01-21 Cantab Biopharmaceuticals Patents Ltd Optimised subcutaneous therapeutic agents
WO2014060401A1 (en) * 2012-10-15 2014-04-24 Novo Nordisk Health Care Ag Coagulation factor vii polypeptides
JP2015533152A (en) 2012-10-15 2015-11-19 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー Factor VII conjugate
JP6566869B2 (en) 2012-12-24 2019-08-28 バイエル・ヘルスケア・エルエルシーＢａｙｅｒ ＨｅａｌｔｈＣａｒｅ ＬＬＣ Short-acting factor VII polypeptide
EP2981282B1 (en) 2013-04-05 2020-11-04 Novo Nordisk Health Care AG Growth hormone compound formulation
WO2015023891A2 (en) 2013-08-14 2015-02-19 Biogen Idec Ma Inc. Factor viii-xten fusions and uses thereof
AU2014336252A1 (en) * 2013-10-15 2016-04-07 Novo Nordisk Health Care Ag Coagulation factor VII polypeptides
EA201890423A1 (en) 2015-08-03 2018-07-31 Биовератив Терапьютикс Инк. SLIGHT PROTEINS OF THE FACTOR IX, METHODS OF THEIR RECEPTION AND APPLICATION
RU2633588C2 (en) * 2015-11-03 2017-10-13 Евгений Федорович Чередников Method for endoscopic treatment of ulcerous gastroduodenal bleeding
KR20260008164A (en) 2016-12-02 2026-01-15 바이오버라티브 테라퓨틱스 인크. Methods of treating hemophilic arthropathy using chimeric clotting factors
MA52630B1 (en) 2018-05-18 2025-07-31 Bioverativ Therapeutics Inc. METHODS OF TREATMENT OF HEMOPHILIA A
CN114728044A (en) 2019-08-15 2022-07-08 介控生化科技公司 Modified factor VII polypeptides for subcutaneous administration and on-demand treatment
AU2020382621A1 (en) 2019-11-13 2022-06-02 Amunix Pharmaceuticals, Inc. Barcoded XTEN polypeptides and compositions thereof, and methods for making and using the same
IL312462A (en) * 2021-10-29 2024-06-01 Sigilon Therapeutics Inc Compositions for cell-based therapies and related methods

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580560A (en) * 1989-11-13 1996-12-03 Novo Nordisk A/S Modified factor VII/VIIa

Family Cites Families (139)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4470461A (en) 1982-09-30 1984-09-11 Phillips Petroleum Company Organic nitro compounds as cosurfactants in enhanced oil recovery processes
US4870008A (en) 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
DE3572982D1 (en) 1984-03-06 1989-10-19 Takeda Chemical Industries Ltd Chemically modified lymphokine and production thereof
US4931373A (en) 1984-05-25 1990-06-05 Zymogenetics, Inc. Stable DNA constructs for expression of α-1 antitrypsin
GB8430252D0 (en) 1984-11-30 1985-01-09 Beecham Group Plc Compounds
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
ZA862768B (en) * 1985-04-17 1986-12-30 Zymogenetics Inc Expression of factor vii and ix activities in mammalian cells
GR860984B (en) 1985-04-17 1986-08-18 Zymogenetics Inc Expression of factor vii and ix activities in mammalian cells
WO1987000056A1 (en) 1985-06-26 1987-01-15 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
ATE93541T1 (en) 1985-07-03 1993-09-15 Genencor Int HYBRID POLYPEPTIDES AND PROCESS FOR THEIR PRODUCTION.
US5679543A (en) 1985-08-29 1997-10-21 Genencor International, Inc. DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
WO1987002670A1 (en) 1985-10-25 1987-05-07 Mackay Vivian L Method of using bar1 for secreting foreign proteins
US5180583A (en) 1985-11-26 1993-01-19 Hedner Ulla K E Method for the treatment of bleeding disorders
GB8601597D0 (en) 1986-01-23 1986-02-26 Wilson R H Nucleotide sequences
JPS63502716A (en) 1986-03-07 1988-10-13 マサチューセッツ・インステチュート・オブ・テクノロジー How to enhance glycoprotein stability
DK122686D0 (en) 1986-03-17 1986-03-17 Novo Industri As PREPARATION OF PROTEINS
US5258288A (en) 1986-07-25 1993-11-02 Genzyme Corporation Vector containing DNA encoding mature human protein S
DK323587D0 (en) 1987-06-25 1987-06-25 Novo Industri As PROTEIN
JP2799316B2 (en) 1987-06-12 1998-09-17 ヘキスト・マリオン・ルセル株式会社 Hybrid human protein C and its genetic engineering method
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US5648254A (en) 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
US4847325A (en) 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
GB8809129D0 (en) 1988-04-18 1988-05-18 Celltech Ltd Recombinant dna methods vectors and host cells
JPH0246296A (en) 1988-08-09 1990-02-15 Hoechst Japan Ltd Hybrid protein c and production thereof
JP2928287B2 (en) * 1988-09-29 1999-08-03 協和醗酵工業株式会社 Novel polypeptide
US5218092A (en) * 1988-09-29 1993-06-08 Kyowa Hakko Kogyo Co., Ltd. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
GB8824591D0 (en) 1988-10-20 1988-11-23 Royal Free Hosp School Med Fractionation process
US5349052A (en) 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
AU4647889A (en) 1988-11-18 1990-06-12 Cetus Corporation Insect signal peptide mediated secretion of recombinant proteins
US5041376A (en) 1988-12-09 1991-08-20 The Board Of Regents Of The University Of Texas System Method for identifying or shielding functional sites or epitopes of proteins that enter the exocytotic pathway of eukaryotic cells, the mutant proteins so produced and genes encoding said mutant proteins
US5047335A (en) 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
JP2989002B2 (en) 1988-12-22 1999-12-13 キリン―アムジエン・インコーポレーテツド Chemically modified granulocyte colony stimulating factor
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
ES2247656T3 (en) 1989-04-19 2006-03-01 Enzon, Inc. A PROCESS TO FORM A MODIFIED POLYPEPTIDE THAT INCLUDES A POLYPEPTIDE AND A POLYCHYLENE OXIDE.
US5122614A (en) 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
EP0400472B1 (en) 1989-05-27 1996-04-03 Sumitomo Pharmaceuticals Company, Limited Process for preparing polyethylene glycol derivatives and modified protein.
US5093317A (en) 1989-06-05 1992-03-03 Cephalon, Inc. Treating disorders by application of insulin-like growth factor
US5077214A (en) 1989-07-07 1991-12-31 The Texas A&M University System Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells
US4973317A (en) 1989-07-14 1990-11-27 Bobrove Arthur M Automatic sheath protection of hypodermic needle
US5023328A (en) 1989-08-04 1991-06-11 The Texas A&M University System Lepidopteran AKH signal sequence
US5225537A (en) 1989-12-29 1993-07-06 Zymogenetics, Inc. Methods for producing hybrid phospholipid-binding proteins
JP2549224B2 (en) 1990-01-26 1996-10-30 イムノ・アクチェンゲゼルシャフト Recombinantly produced blood factor, method for expressing the blood factor, and vaccinia virus recombinant used in the method
WO1991011514A1 (en) 1990-01-29 1991-08-08 Zymogenetics, Inc. Anticoagulant proteins
US5219564A (en) 1990-07-06 1993-06-15 Enzon, Inc. Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon
US5583107A (en) 1990-09-04 1996-12-10 Cor Therapeutics, Inc. Agents affecting thrombosis and hemostasis
US5531916A (en) 1990-10-03 1996-07-02 E. I. Du Pont De Nemours And Company Hydrofluorocarbon cleaning compositions
EP0513332A4 (en) 1990-11-14 1993-03-17 Cargill, Incorporated Conjugates of poly(vinylsaccharide) with proteins for the stabilization of proteins
IE914102A1 (en) 1990-11-26 1992-06-03 Genetics Inst Expression of pace in host cells and methods of use thereof
US5833982A (en) 1991-02-28 1998-11-10 Zymogenetics, Inc. Modified factor VII
EP0575464B1 (en) 1991-02-28 2004-08-11 Zymogenetics, Inc. Modified factor vii
US5817788A (en) 1991-02-28 1998-10-06 Zymogenetics, Inc. Modified factor VII
US5788965A (en) 1991-02-28 1998-08-04 Novo Nordisk A/S Modified factor VII
US5861374A (en) * 1991-02-28 1999-01-19 Novo Nordisk A/S Modified Factor VII
WO1992016555A1 (en) 1991-03-18 1992-10-01 Enzon, Inc. Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US5504064A (en) 1991-04-10 1996-04-02 Oklahoma Medical Research Foundation Treatment of bleeding with modified tissue factor in combination with an activator of FVII
DE4113487C1 (en) 1991-04-25 1992-11-05 Fleischgrosshandel Hans-Werner & Bernd Meixner Gmbh, 6301 Wettenberg, De
US5281698A (en) 1991-07-23 1994-01-25 Cetus Oncology Corporation Preparation of an activated polymer ester for protein conjugation
ZA933926B (en) 1992-06-17 1994-01-03 Amgen Inc Polyoxymethylene-oxyethylene copolymers in conjuction with blomolecules
AU5006993A (en) 1992-08-21 1994-03-15 Enzon, Inc. Novel attachment of polyalkylene oxides to bio-effecting substances
US5382657A (en) 1992-08-26 1995-01-17 Hoffmann-La Roche Inc. Peg-interferon conjugates
NZ250375A (en) 1992-12-09 1995-07-26 Ortho Pharma Corp Peg hydrazone and peg oxime linkage forming reagents and protein derivatives
US5298643A (en) 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5321095A (en) 1993-02-02 1994-06-14 Enzon, Inc. Azlactone activated polyalkylene oxides
IL104734A0 (en) 1993-02-15 1993-06-10 Univ Bar Ilan Bioactive conjugates of cellulose with amino compounds
DK38293D0 (en) 1993-03-31 1993-03-31 Novo Nordisk As PREPARATION OF PROTEINS
US5871384A (en) * 1993-04-20 1999-02-16 Kichijo; Hiroshi Block assembly and devices formed thereby
JPH0720127A (en) 1993-05-07 1995-01-24 Eisai Co Ltd Measuring methods and measuring reagents for various PIVKA
PT699075E (en) 1993-05-21 2002-05-31 Novo Nordisk As MODIFIED FACTOR VII FOR INHIBITION OF VASCULAR RESTENOSE AND PLATELET DEPOSITION
WO1994028024A1 (en) 1993-06-01 1994-12-08 Enzon, Inc. Carbohydrate-modified polymer conjugates with erythropoietic activity
AU7113594A (en) 1993-06-21 1995-01-17 Enzon, Inc. Site specific synthesis of conjugated peptides
GB9317618D0 (en) 1993-08-24 1993-10-06 Royal Free Hosp School Med Polymer modifications
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5951974A (en) 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
NZ276943A (en) 1993-11-10 1998-02-26 Schering Corp Substituted For Alpha-interferon conjugated to a non-antigenic polymer (preferably a polyalkylene oxide) and its preparation
US5446090A (en) 1993-11-12 1995-08-29 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
DE4343591A1 (en) 1993-12-21 1995-06-22 Evotec Biosystems Gmbh Process for the evolutionary design and synthesis of functional polymers based on shape elements and shape codes
US6117679A (en) 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5473034A (en) 1994-03-18 1995-12-05 Hyogo Prefectural Government Method for producing protein-synthetic polymer conjugate and said conjugate produced thereby
US5629384A (en) 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
AU2826495A (en) 1994-06-02 1996-01-04 Enzon, Inc. Method of solubilizing substantially water insoluble materials
US5730990A (en) 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
EP1559776A3 (en) 1994-06-30 2006-01-11 Novozymes Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic Fusarium expression system and promoters and terminators for use therein
US5650234A (en) 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5824784A (en) 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
DE4437604A1 (en) 1994-10-21 1996-04-25 Basf Ag Conjugates of a poly or oligopeptide and a low molecular weight lipophilic compound
CA2204726A1 (en) 1994-11-09 1996-12-27 Robin E. Offord Functionalized polymers for site-specific attachment
US5738846A (en) 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5932462A (en) 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
AU5893696A (en) 1995-06-07 1996-12-30 Novo Nordisk A/S Modification of polypeptides
US5672662A (en) 1995-07-07 1997-09-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
WO1997007205A1 (en) 1995-08-11 1997-02-27 Novo Nordisk A/S Method for preparing polypeptide variants
DE19531637A1 (en) 1995-08-28 1997-03-06 Immuno Ag Pharmaceutical composition for the treatment of blood coagulation disorders, method for the production thereof and their use
US5747639A (en) 1996-03-06 1998-05-05 Amgen Boulder Inc. Use of hydrophobic interaction chromatography to purify polyethylene glycols
TW517067B (en) 1996-05-31 2003-01-11 Hoffmann La Roche Interferon conjugates
SI0964702T1 (en) 1996-08-02 2007-02-28 Ortho Mcneil Pharm Inc Polypeptides having a single covalently bound n-terminal water-soluble polymer
US5837843A (en) 1996-11-08 1998-11-17 Oklahoma Medical Research Foundation Modified protein C
ATE321846T1 (en) 1996-11-08 2006-04-15 Oklahoma Med Res Found USE OF A MODIFIED PROTEIN-C
EP0954589A1 (en) 1997-01-24 1999-11-10 Novo Nordisk A/S Synthetic leader peptide sequences
WO1998032466A1 (en) 1997-01-29 1998-07-30 Polymasc Pharmaceuticals Plc Pegylation process
CA2279986A1 (en) 1997-02-06 1998-08-13 Novo Nordisk A/S Polypeptide-polymer conjugates having added and/or removed attachment groups
US6323322B1 (en) 1997-04-30 2001-11-27 Enzon, Inc. Single-chain antigen-binding proteins capable of glycosylation, production and uses thereof
EP0921131B1 (en) 1997-06-06 2010-05-26 Kyowa Hakko Kirin Co., Ltd. Chemically modified polypeptides
US6475725B1 (en) 1997-06-20 2002-11-05 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
AT407255B (en) 1997-06-20 2001-02-26 Immuno Ag RECOMBINANT CELL CLONE WITH INCREASED STABILITY IN SERUM- AND PROTEIN-FREE MEDIUM AND METHOD FOR OBTAINING THE STABLE CELL CLONE
DE69838552T2 (en) * 1997-07-14 2008-05-21 Bolder Biotechnology, Inc., Louisville DERIVATIVES OF GROWTH HORMONE AND RELATED PROTEINS
WO1999003498A1 (en) 1997-07-18 1999-01-28 Novo Nordisk A/S USE OF FVIIa OR FVIIai FOR THE TREATMENT OF ADVERSE CONDITIONS RELATED TO THE FVIIa MEDIATED INTRACELLULAR SIGNALLING PATHWAY
US6017882A (en) 1997-10-23 2000-01-25 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US7247708B2 (en) 1997-10-23 2007-07-24 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US6693075B1 (en) 1997-10-23 2004-02-17 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US6747003B1 (en) 1997-10-23 2004-06-08 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US5981709A (en) 1997-12-19 1999-11-09 Enzon, Inc. α-interferon-polymer-conjugates having enhanced biological activity and methods of preparing the same
US5985263A (en) 1997-12-19 1999-11-16 Enzon, Inc. Substantially pure histidine-linked protein polymer conjugates
AU762621B2 (en) 1998-04-28 2003-07-03 Merck Serono Sa Polyol-IFN-beta conjugates
AT408613B (en) 1998-06-17 2002-01-25 Immuno Ag PHARMACEUTICAL FACTOR VII PREPARATION
EP1124843B1 (en) 1998-10-30 2004-04-07 Novozymes A/S Low allergenic protein variants
CA2348822A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Glycosylated proteins having reduced allergenicity
WO2000028065A1 (en) 1998-11-06 2000-05-18 Novo Nordisk A/S Method for the production of fvii
ATE465459T1 (en) 1999-01-19 2010-05-15 Maxygen Inc THROUGH OLIGONUCLEOTIDE-MEDIATED NUCLEIC ACID RECOMBINATION
AU3629800A (en) 1999-03-16 2000-10-04 Children's Hospital Of Philadelphia, The Enhanced gamma-carboxylation of recombinant vitamin k-dependent clotting factors
US6432826B1 (en) * 1999-11-29 2002-08-13 Applied Materials, Inc. Planarized Cu cleaning for reduced defects
CN100488562C (en) 2000-02-11 2009-05-20 拜耳医药保健有限公司 Coagulation factor VII or VIIa-like molecules
US7220837B1 (en) 2000-04-28 2007-05-22 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
ATE485371T1 (en) 2000-05-03 2010-11-15 Novo Nordisk Healthcare Ag VARIANTS OF THE HUMAN COAGULATION FACTOR VII
US6423826B1 (en) 2000-06-30 2002-07-23 Regents Of The University Of Minnesota High molecular weight derivatives of vitamin K-dependent polypeptides
US7160540B2 (en) 2000-06-30 2007-01-09 Regents Of The University Of Minnesota Methods for detecting activity of clottings factors
US20030211094A1 (en) 2001-06-26 2003-11-13 Nelsestuen Gary L. High molecular weight derivatives of vitamin k-dependent polypeptides
US7176288B2 (en) 2000-09-13 2007-02-13 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
JP4361728B2 (en) 2000-09-13 2009-11-11 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Human coagulation factor VII mutant
BR0114374A (en) 2000-10-02 2003-12-30 Novo Nordisk As Preparation comprising a plurality of factor vii polypeptides, methods for determining the pattern of factor vii glycoform and factor vii-related polypeptides, and for producing said preparation, pharmaceutical formulation, methods for treating a factor responsive syndrome. vii, for the prevention of unwanted bleeding, for the prevention of unwanted blood clotting, and for the prevention of tissue factor mediated reactions, and the use of the preparation.
AU2002218029A1 (en) 2000-11-09 2002-05-21 The Scripps Research Institute Modified factor viia
MXPA04010781A (en) 2002-04-30 2005-03-07 Maxygen Holdings Ltd FACTOR VII OR VIIa POLYPEPTIDE VARIANTS.
CA2502162C (en) 2002-09-30 2014-04-15 Bayer Healthcare Llc Fvii or fviia variants having increased clotting activity
ES2327044T3 (en) 2003-03-20 2009-10-23 Bayer Healthcare Llc VARIATIONS OF FVII OR FVIIA.
MXPA05013769A (en) 2003-06-19 2006-03-08 Maxygen Holdings Ltd FACTOR VII OR VIIa GLA DOMAIN VARIANTS.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580560A (en) * 1989-11-13 1996-12-03 Novo Nordisk A/S Modified factor VII/VIIa

Also Published As

Publication number Publication date
CA2739933A1 (en) 2001-08-16
EP2319541A1 (en) 2011-05-11
PL206148B1 (en) 2010-07-30
US7598056B2 (en) 2009-10-06
PT1257295E (en) 2009-07-16
US7427592B2 (en) 2008-09-23
PL362856A1 (en) 2004-11-02
CA2397347A1 (en) 2001-08-16
US20070243588A1 (en) 2007-10-18
US20060228782A1 (en) 2006-10-12
US20070142280A1 (en) 2007-06-21
US7517974B2 (en) 2009-04-14
US20030096338A1 (en) 2003-05-22
US7442524B2 (en) 2008-10-28
NO20023804D0 (en) 2002-08-09
US20060252689A1 (en) 2006-11-09
NO20023804L (en) 2002-09-25
US20060240524A1 (en) 2006-10-26
PL204285B1 (en) 2009-12-31
IL199497A0 (en) 2011-07-31
US20060019336A1 (en) 2006-01-26
JP2003521930A (en) 2003-07-22
CN1400910A (en) 2003-03-05
US7414022B2 (en) 2008-08-19
ATE428445T1 (en) 2009-05-15
JP2005270110A (en) 2005-10-06
US8084591B2 (en) 2011-12-27
US20120171765A1 (en) 2012-07-05
US20090023635A1 (en) 2009-01-22
DE60138364D1 (en) 2009-05-28
WO2001058935A2 (en) 2001-08-16
US7371543B2 (en) 2008-05-13
US7511024B2 (en) 2009-03-31
AU2006200448B2 (en) 2009-11-12
EP1257295B1 (en) 2009-04-15
NZ521257A (en) 2004-10-29
IL150692A0 (en) 2003-02-12
EP1982732A3 (en) 2011-06-08
US7754682B2 (en) 2010-07-13
DK1257295T3 (en) 2009-08-10
EP1982732A2 (en) 2008-10-22
AU2010200296A1 (en) 2010-02-18
JP4509846B2 (en) 2010-07-21
WO2001058935A3 (en) 2001-11-29
IL199497A (en) 2013-08-29
US20060258585A1 (en) 2006-11-16
RU2002124129A (en) 2004-03-20
US20060252690A1 (en) 2006-11-09
EP1257295A2 (en) 2002-11-20
US20060252127A1 (en) 2006-11-09
AU3153501A (en) 2001-08-20
ES2325877T3 (en) 2009-09-23
CY1109614T1 (en) 2014-08-13
AU783512B2 (en) 2005-11-03
AU2006200448A1 (en) 2006-03-02
RU2278123C2 (en) 2006-06-20
US20060240525A1 (en) 2006-10-26
CA2397347C (en) 2011-07-12
US6806063B2 (en) 2004-10-19

Similar Documents

Publication Publication Date Title
CN100488562C (en) Coagulation factor VII or VIIa-like molecules
CA2502162C (en) Fvii or fviia variants having increased clotting activity
US20060270001A1 (en) Factor VII or VIIa Polypeptide Variants
CN101503726A (en) Factor VII or VIIa-like molecules

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
ASS Succession or assignment of patent right

Free format text: FORMER OWNER: MAXIGEN HOLDINGS CO., LTD.

Effective date: 20050408

Owner name: MAXIGEN HOLDINGS CO., LTD.

Free format text: FORMER OWNER: MARKCHIGAN CO.,LTD.

C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20050408

Address after: Cayman Islands

Applicant after: Maxygen Holdings Ltd.

Address before: Holm Hess, Denmark

Applicant before: Maxygen Inc.

Co-applicant before: Maxygen Holdings Ltd.

C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20090520

Termination date: 20160212

CF01 Termination of patent right due to non-payment of annual fee